Tubular nanostructure targeted to cell membrane

ABSTRACT

Devices, compositions, and methods are described which provide a tubular nanostructure or a composite tubular nanostructure targeted to a lipid bilayer membrane. The tubular nanostructure includes a hydrophobic surface region flanked by two hydrophilic surface regions. The tubular nanostructure is configured to interact with a lipid bilayer membrane and form a pore in the lipid bilayer membrane. The tubular nanostructure may be targeted by including at least one ligand configured to bind to one or more cognates on the lipid bilayer membrane of a target cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims the benefit of theearliest available effective filing date(s) from the following listedapplication(s) (the “Related Applications”) (e.g., claims earliestavailable priority dates for other than provisional patent applicationsor claims benefits under 35 USC § 119(e) for provisional patentapplications, for any and all parent, grandparent, great-grandparent,etc. applications of the Related Application(s)). All subject matter ofthe Related Applications and of any and all parent, grandparent,great-grandparent, etc. applications of the Related Applications isincorporated herein by reference to the extent such subject matter isnot inconsistent herewith.

REGARDING PRIORITY BENEFITS

The present application constitutes a continuation of U.S. patentapplication Ser. No. 12/322,367, entitled Tubular Nanostructure Targetedto Cell Membrane, naming Mahalaxmi Gita Bangera, Ed Harlow, Roderick A.Hyde, Muriel Y. Ishikawa, Edward K. Y. Jung, Eric C. Leuthardt, NathanP. Myhrvold, Dennis J. Rivet, Elizabeth A. Sweeney, Clarence T.Tegreene, Lowell L. Wood, Jr., Victoria Y. H. Wood as inventors, filed30 Jan. 2009, which is currently co-pending or is an application ofwhich a currently co-pending application is entitled to the benefit ofthe filing date, and which is a continuation of U.S. patent applicationSer. No. 12/283,907, entitled Tubular Nanostructure Targeted to CellMembrane, naming Mahalaxmi Gita Bangera, Ed Harlow, Roderick A. Hyde,Muriel Y. Ishikawa, Edward K. Y. Jung, Eric C. Leuthardt, Nathan P.Myhrvold, Dennis J. Rivet, Elizabeth A. Sweeney, Clarence T. Tegreene,Lowell L. Wood, Jr. and Victoria Y. H. Wood as inventors, filed 15 Sep.2008.

For purposes of the USPTO extra-statutory requirements, the presentapplication constitutes a continuation-in-part of United States PatentApplication No. Ser. No. 12/283,907, entitled Tubular NanostructureTargeted To Cell Membrane, naming Mahalaxmi Gita Bangera, Ed Harlow,Roderick A. Hyde, Muriel Y. Ishikawa, Edward K. Y. Jung, Jordin T. Kare,Eric C. Leuthardt, Nathan P. Myhrvold, Dennis J. Rivet, Elizabeth A.Sweeney, Clarence T. Tegreene, Lowell L. Wood, Jr. and Victoria Y. H.Wood as inventors, filed 15 Sep. 2008, which is currently co-pending, oris an application of which a currently co-pending application isentitled to the benefit of the filing data.

The United States Patent Office (USPTO) has published a notice to theeffect that the USPTO's computer programs require that patent applicantsreference both a serial number and indicate whether an application is acontinuation or continuation-in-part. Stephen G. Kunin, Benefit ofPrior-Filed Application, USPTO Official Gazette Mar. 18, 2003. Thepresent Applicant Entity (hereinafter “Applicant”) has provided above aspecific reference to the application(s) from which priority is beingclaimed as recited by statute. Applicant understands that the statute isunambiguous in its specific reference language and does not requireeither a serial number or any characterization, such as “continuation”or “continuation-in-part,” for claiming priority to U.S. patentapplications. Notwithstanding the foregoing, Applicant understands thatthe USPTO's computer programs have certain data entry requirements, andhence Applicant is designating the present application as acontinuation-in-part of its parent applications as set forth above, butexpressly points out that such designations are not to be construed inany way as any type of commentary and/or admission as to whether or notthe present application contains any new matter in addition to thematter of its parent application(s).

All subject matter of the Related Applications and of any and allparent, grandparent, great-grandparent, etc. applications of the RelatedApplications is incorporated herein by reference to the extent suchsubject matter is not inconsistent herewith.

SUMMARY

Devices, compositions, and methods described herein provide a tubularnanostructure targeted to a lipid bilayer membrane. The targeted tubularnanostructure can have a hydrophobic surface region flanked by twohydrophilic surface regions. The tubular nanostructure is configured tointeract with a lipid bilayer membrane and form a pore in the lipidbilayer membrane. The tubular nanostructure may be targeted by includingat least one ligand configured to bind to one or more cognates on thelipid bilayer membrane of a target cell, for example, on a tumor cell,an infected cell, or a diseased cell in a subject. The tubularnanostructure can form a pore in the lipid bilayer membrane which canpermit transit or translocation of at least one compound across themembrane and cause cell death of the target cell.

Devices, compositions, and methods described herein provide a tubularnanostructure targeted to a lipid bilayer membrane. The targeted tubularnanostructure can have a hydrophobic surface region flanked by twohydrophilic surface regions. The tubular nanostructure is configured tointeract with a lipid bilayer membrane and form a pore in the lipidbilayer membrane. The tubular nanostructure may be targeted by includingat least one ligand configured to bind to one or more cognates on thelipid bilayer membrane of a target cell, for example, on a tumor cell,an infected cell, or a diseased cell in a subject. The tubularnanostructure can form a pore in the lipid bilayer membrane which canpermit transit or translocation of at least one compound across themembrane and cause cell death of the target cell.

A tubular nanostructure is provided which includes a hydrophobic surfaceregion flanked by two hydrophilic surface regions configured to form apore in a lipid bilayer membrane, and at least one ligand configured tobind one or more cognates on the membrane. The nanostructure includes,but is not limited to, one or more of a carbon nanotube, cyclic peptidenanotube, crown ether nanotube, polymer nanotube, polymer/carbonnanotube, DNA nanotube, or inorganic nanotube. The inorganic nanotubefurther includes, but is not limited to, a boron nitride nanotube. Thepolymer nanotube includes, but is not limited to, polystyrene,polytetrafluoroethylene, polymethylmethacrylate, polyaniline, orpoly-L-lactide/palladium acetate. The polymer/carbon nanotube includes,but is not limited to, a polyaniline/carbon nanotube. The hydrophobicsurface region includes, but is not limited to, a single wall carbonnanotube surface region. The hydrophilic surface region includes, but isnot limited to, one or more of amines, amides, charged or polar aminoacids, alcohols, carboxylic groups, oxides, ester groups, ether groups,or ester-ether groups, ketones, aldehydes, or derivatives thereof. Theone or more cognates include, but are not limited to, one or more cellsurface receptors or cell surface markers in the lipid bilayer membrane.The one or more cognates include, but are not limited to, at least oneof a protein, a carbohydrate, a glycoprotein, a glycolipid, asphingolipid, a glycerolipid or a metabolite thereof. One or both of thehydrophilic surface regions may be at the end of the nanostructure. Thenanostructure may have a length of about 1 nm to about 1500 nm, or alength of about 20 Å to about 40 Å. The nanostructure may have adiameter of about 0.5 nm to about 5 nm or a diameter of about 5 Å toabout 20 Å. The at least one ligand includes, but is not limited to, atleast a portion of an antibody, antibody-coated liposome,polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein,small chemical compound, carbohydrate, lipid, toxin, pore-forming toxin,or lectin. The at least one ligand includes a therapeutic compoundconfigured to affect a cell or process, or to treat at least one of adisease, condition, or symptom. The nanostructure may further include atleast one second ligand configured to bind one or more cognates on thelipid bilayer membrane. The tubular nanostructure induces cell death.

The nanostructure may further include one or more elements to controltransport of molecules through the tubular nanostructure. In one aspect,the one or more elements are on the extracellular end of thenanostructure. In a further aspect, the one or more elements are on thecytoplasmic end of the nanostructure. The one or more elements mayinclude a hydrophilic inner liner of the tubular nanostructure. The oneor more elements further includes at least one second ligand configuredto reversibly bind a cognate of interest, wherein the cognate ofinterest passes through the pore. The at least one second ligandincludes, but is not limited to, a monospecific antibody or a bispecificantibody. The one or more elements may reversibly block the pore. Theone or more elements includes, but is not limited to, a magnetic entityor a molecular entity. The molecular entity includes, but is not limitedto, at least a portion of a carbon nanostructure, polynucleotide,polypeptide, antibody, receptor, glycoprotein, lipid, polysaccharide, orpolymer. The one or more elements may include a charged group. The oneor more elements may be passive or active. In one aspect, the porepermits transit or translocation of at least one compound across themembrane. The one or more active elements includes, but is not limitedto, at least one of an ATPase transport element, Na⁺K⁺ ATPase, H⁺K⁺ATPase, or Ca²⁺ ATPase. The one or more active elements furtherincludes, but is not limited to, at least one of an ABC transporterelement, CFTR transporter, TAP transporter, or liver cell transporter.The one or more active elements further includes, but is not limited to,at least one of a symport pump, Na⁺/iodide transporter, E. colipermease, or an antiport pump.

The nanostructure may further include a marker attached to thenanostructure. The marker includes, but is not limited to, a fluorescentmarker, a radioactive marker, quantum dot, metal, or magnetic resonanceimaging marker. The marker may be activated by anchoring in themembrane. The marker may be activated by a ligand reaction. The markermay be activated by interaction with a hydrophobic medium. Thehydrophobic surface region may be extended in diameter. The hydrophobicsurface region may be extended in diameter by a disk, a stub, or agraphene sheet.

A composite tubular nanostructure includes two or more nanotubes whereinat least one nanotube includes a hydrophobic surface region, eachhydrophobic surface region flanked by two hydrophilic surface regionsconfigured to form a pore in a lipid bilayer membrane. The compositetubular nanostructure may further include at least one ligand configuredto bind one or more cognates on the lipid bilayer membrane. The two ormore ligands may be configured to bind to the one or more cognates onthe lipid bilayer membrane. The composite tubular nanostructure mayfurther include three or more nanotubes. The composite tubularnanostructure may further include at least one nanotube includes acompletely hydrophobic surface region. The at least one nanotubeincluding the completely hydrophobic surface region may be surrounded byat least six nanotubes including the hydrophobic surface region flankedby two hydrophilic surface regions configured to form the pore in thelipid bilayer membrane. The at least two of the nanotubes may havedifferent diameters. The at least two of the nanotubes may havedifferent lengths. The nanotubes may be substantially parallel. Thenanotubes may be substantially orthogonal. The composite tubularnanostructure includes, but is not limited to, at least one of the twoor more nanotubes is a carbon nanotube, cyclic peptide nanotube, crownether nanotube, polymer nanotube, polymer/carbon nanotube, DNA nanotube,or inorganic nanotube. The inorganic nanotube further includes, but isnot limited to, a boron nitride nanotube. The polymer nanotube includes,but is not limited to, polystyrene, polytetrafluoroethylene,polymethylmethacrylate, polyaniline, or poly-L-lactide/palladiumacetate. The polymer/carbon nanotube includes, but is not limited to, apolyaniline/carbon nanotube. The hydrophobic surface region includes,but is not limited to, a single wall carbon nanotube surface region. Thehydrophilic surface region includes, but is not limited to, one or moreof amines, amides, charged or polar amino acids, alcohols, carboxylicgroups, oxides, ester groups, ether groups, or ester-ether groups,ketones, aldehydes, or derivatives thereof. The one or more cognatesinclude, but are not limited to, one or more cell surface receptors orcell surface markers in the lipid bilayer membrane. The one or morecognates include, but are not limited to, at least one of a protein, acarbohydrate, a glycoprotein, a glycolipid, a sphingolipid, aglycerolipid or a metabolite thereof. One or both of the hydrophilicsurface regions may be at the end of the nanostructure. Thenanostructure may have a length of about 1 nm to about 1500 nm, or alength of about 20 Å to about 40 Å. The nanostructure may have adiameter of about 0.5 nm to about 5 nm or a diameter of about 5 Å toabout 20 Å. The at least one ligand includes, but is not limited to, atleast a portion of an antibody, antibody-coated liposome,polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein,small chemical compound, carbohydrate, lipid, toxin, pore-forming toxin,or lectin. The at least one ligand includes a therapeutic compoundconfigured to affect a cell or process, or to treat at least one of adisease, condition, or symptom. The nanostructure may further include atleast one second ligand configured to bind one or more cognates on thelipid bilayer membrane. The two or more ligands may be configured tobind to the one or more cognates on the lipid bilayer membrane. Thecomposite tubular nanostructure may further include a therapeuticcomposition to treat a disease, symptom, or condition. The therapeuticcomposition includes, but is not limited to, a cytotoxic agent orantimicrobial agent.

The composite tubular nanostructure may further include one or moreelements to control transport of molecules through the tubularnanostructure. In one aspect, the one or more elements are on theextracellular end of the nanostructure. In a further aspect, the one ormore elements are on the cytoplasmic end of the nanostructure. The oneor more elements may include a hydrophilic inner liner of the tubularnanostructure. The one or more elements further includes a ligand. Theone or more elements may include a charged group. The one or moreelements may be passive or active. The one or more elements may havedifferent transport properties. The one or more active elementsincludes, but is not limited to, at least one of an ATPase transportelement, Na⁺K⁺ ATPase, H⁺K⁺ ATPase, or Ca²⁺ ATPase. The one or moreactive elements further includes, but is not limited to, at least one ofan ABC transporter element, CFTR transporter, TAP transporter, or livercell transporter. The one or more active elements further includes, butis not limited to, at least one of a symport pump, Na⁺/iodidetransporter, E. coli permease, or an antiport pump. In one aspect, thepore permits transit or translocation of at least one compound acrossthe membrane. The nanostructure may further include a marker attached tothe nanostructure. The marker includes, but is not limited to, afluorescent marker, a radioactive marker, quantum dot, metal, ormagnetic resonance imaging marker. The marker may be activated byanchoring in the membrane. The marker may be activated by a ligandreaction. The marker may be activated by interaction with a hydrophobicmedium. In one aspect, the cognate includes one or more cell surfacereceptors or cell surface markers on a neoplastic cell or an infectedcell

A method for inserting a tubular nanostructure into a lipid bilayermembrane is provided which includes applying to a lipid bilayermembrane, a tubular nanostructure including a hydrophobic surface regionflanked by two hydrophilic surface regions configured to form a pore inthe lipid bilayer membrane and including at least one ligand configuredto bind one or more cognates on the membrane, under conditions and fortime sufficient to allow the nanostructure to penetrate the membrane. Inone aspect, one or both of the hydrophilic regions of the nanostructureis located substantially at an end of the tubular nanostructure. Thetubular nanostructure includes, but is not limited to, a carbonnanotube, cyclic peptide nanotube, crown ether nanotube, polymernanotube, polymer/carbon nanotube, DNA nanotube, or inorganic nanotube.The hydrophilic regions of the tubular body include, but are not limitedto, one or more of amines, amides, charged or polar amino acids,alcohols, carboxylic groups, ester groups, oxides, ether groups,ester-ether groups, ketones, aldehydes, or derivatives thereof. In oneaspect, the tubular nanostructure is assisted in crossing the membranecore by lipid molecules from the membrane. In a further aspect, thelipid molecules assisting the tubular nanostructure in crossing themembrane undergo lipid translocation across a bilayer leaflet.

A method for providing a stable pore in a lipid bilayer membrane isprovided which includes positioning across a lipid bilayer membrane atubular nanostructure including a hydrophobic surface region flanked bytwo hydrophilic surface regions configured to form a pore in the lipidbilayer membrane and including at least one ligand configured to bindone or more cognates on the membrane. The one or more of the hydrophilicregions of the tubular nanostructure may be located at an end of atubular nanostructure. In one aspect, the both hydrophilic regions maybe located at opposite ends of a tubular nanostructure. In one aspect,positioning the tubular nanostructure across the lipid bilayer membraneinduces cell death. The lipid bilayer membrane may be on a neoplasticcell or an infected cell.

A method for inserting a tubular nanostructure into a lipid bilayermembrane is provided which includes applying to a lipid bilayer membranea composite tubular nanostructure including two or more nanotubeswherein at least one nanotube includes a hydrophobic surface regionflanked by two hydrophilic surface regions configured to form a pore ina lipid bilayer membrane, under conditions and for time sufficient toallow the composite nanostructure to penetrate the membrane. Thehydrophobic region of at least one of the two or more nanotubes may belocated at the ends of a tubular body. The hydrophilic regions may belocated at the ends of the at least one nanotube. The tubularnanostructure may include a composite nanostructure of three or morenanotubes. The composite tubular nanostructure may further include atleast one nanotube includes a completely hydrophobic surface region. Theat least one nanotube including the completely hydrophobic surfaceregion may be surrounded by at least six nanotubes including thehydrophobic surface region flanked by two hydrophilic surface regionsconfigured to form the pore in the lipid bilayer membrane. The at leasttwo of the nanotubes may have different diameters. The at least two ofthe nanotubes may have different lengths. The tubular nanostructure maybe assisted in crossing the membrane core by lipid molecules from themembrane. The lipid molecules assisting the tubular nanostructure incrossing the membrane may undergo lipid translocation across a bilayerleaflet.

A method for providing a pore in a lipid bilayer membrane includespositioning across a lipid bilayer membrane a composite tubularnanostructure including two or more nanotubes wherein at least onenanotube includes a hydrophobic surface region flanked by twohydrophilic surface regions configured to form a pore in a lipid bilayermembrane. The hydrophilic regions may be located at opposite ends of theat least one nanotubes. The tubular nanostructure may be positionedacross the lipid bilayer membrane induces cell death. The lipid bilayermembrane may be on a neoplastic cell or an infected cell. The tubularnanostructure may be assisted in crossing the membrane core by lipidmolecules from the membrane. The lipid molecules assisting the tubularnanostructure in crossing the membrane may undergo lipid translocationacross a bilayer leaflet.

A method for disrupting a lipid bilayer membrane of a cell is providedwhich includes contacting the cell with at least one tubularnanostructure including a hydrophobic surface region flanked by twohydrophilic surface regions configured to form a pore in the lipidbilayer membrane and including at least one ligand configured to bindone or more cognates on the lipid bilayer membrane of the cell. Two ormore ligands may be configured to bind to the one or more cognates onthe lipid bilayer membrane. The at least one tubular nanostructure maybe positioned across the lipid bilayer membrane induces cell death. Thecell may be a neoplastic cell or an infected cell.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C and 1D depict a diagrammatic view of one aspect of anexemplary embodiment of a tubular nanostructure and a method forinserting a tubular nanostructure into a lipid bilayer membrane of acell.

FIGS. 2A, 2B, 2C, 2D, and 2E depict a diagrammatic view of one aspect ofan exemplary embodiment of a tubular nanostructure and a method forinserting a tubular nanostructure into a lipid bilayer membrane of acellular organelle.

FIG. 3 depicts a logic flowchart of a method for inserting a tubularnanostructure into a lipid bilayer membrane.

FIG. 4 depicts a logic flowchart of a method for providing a stable porein a lipid bilayer membrane.

FIG. 5 depicts a logic flowchart of a method for inserting a tubularnanostructure into a lipid bilayer membrane.

FIG. 6 depicts a logic flowchart of a method for providing a pore in alipid bilayer membrane.

FIG. 7 depicts a logic flowchart of a method for disrupting a lipidbilayer membrane of a cell.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

The present application uses formal outline headings for clarity ofpresentation. However, it is to be understood that the outline headingsare for presentation purposes, and that different types of subjectmatter may be discussed throughout the application (e.g., method(s) maybe described under composition heading(s) and/or kit headings; and/ordescriptions of single topics may span two or more topic headings).Hence, the use of the formal outline headings is not intended to be inany way limiting.

Devices, compositions, and methods described herein provide a tubularnanostructure targeted to a lipid bilayer membrane. The targeted tubularnanostructure can have a hydrophobic surface region flanked by twohydrophilic surface regions. The tubular nanostructure is configured tointeract with a lipid bilayer membrane and form a pore in the lipidbilayer membrane. The tubular nanostructure may be targeted by includingat least one ligand configured to bind to one or more cognates on thelipid bilayer membrane of a target cell, for example, on a tumor cell,an infected cell, or a diseased cell in a subject. The tubularnanostructure can form a pore in the lipid bilayer membrane which canpermit transit or translocation of at least one compound across themembrane and cause cell death of the target cell.

At least one ligand includes a compound that binds a cognate and can beat least a portion of an antibody, antibody-coated liposome,polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein,carbohydrate, lipid, toxin, lectin, pore-forming toxin, small chemicalcompound, or any combination thereof. In one aspect, the ligand can be atherapeutic compound configured to affect a cell or process or to treatat least one of a disease, condition, or symptom

One or more cognates can be associated with a target cell or organelleand may include, but is not limited to, at least one of a protein, acarbohydrate, a glycoprotein, a glycolipid, a sphingolipid, aglycerolipid, or metabolites thereof. The cognate can be a cell surfacereceptor or a cell surface marker on the lipid bilayer membrane of atarget cell, for example, on a tumor cell, an infected cell, or adiseased cell in a subject, or on a bacterial cell or a parasite cell.

Ligands can be targeted to cognates which are associated with lipidbilayer membranes of target cells and/or organelles. A target cell mayinclude a tumor cell and/or other diseased cell type in a mammaliansubject. A target cell may also include a pathogen, e.g., bacteria,fungi, and/or parasites. In some instances, the tubular nanostructuresmay be designed to target a specific cellular organelle, e.g., themitochondria. The tubular nanostructure can include a surface regionconfigured to pass through a lipid bilayer membrane of a cell, ahydrophobic surface region flanked by two hydrophilic surface regionsconfigured to form a pore in a lipid bilayer membrane of a cellularorganelle, and at least one ligand configured to bind one or morecognates on the lipid bilayer membrane of the cellular organelle.

The tubular nanostructure includes, but is not limited to, one or moreof a carbon nanotube, cyclic peptide nanotube, crown ether nanotube,polymer nanotube, polymer/carbon nanotube, DNA nanotube, or inorganicnanotube. The inorganic nanotube can include a boron nitride nanotube.The polymer nanotube can include polystyrene, polytetrafluoroethylene,polymethylmethacrylate, polyaniline, or poly-L-lactide/palladiumacetate. The polymer/carbon nanotube can include a polyaniline/carbonnanotube. A single wall carbon nanotube can have a hydrophobic surfaceregion at least a portion of, or all of, the surface structure of thetubular nanostructure.

A hydrophobic surface region of a tubular nanostructure includes atubular nanostructure with a carbon surface structure and/or a linkermolecule having a hydrophobic portion adsorbed onto the tubularnanostructure, e.g., a phospholipid. A hydrophobic polymer refers to anypolymer resistant to wetting, or not readily wet, by water, i.e., havinga lack of affinity for water. A hydrophobic polymer typically will havea surface free energy of about 40 dynes/cm (10⁻⁵ Newtons/cm or N/cm) orless. Examples of hydrophobic polymers include, by way of illustrationonly, polylactide, polylactic acid, polyolefins, such as poylethylene,poly(isobutene), poly(isoprene), poly(4-methyl-1-pentene),polypropylene, ethylene-propylene copolymers, andethylenepropylene-hexadiene copolymers; ethylene-vinyl acetatecopolymers; styrene polymers, such as poly(styrene), poly(2-methylstyrene), styrene-acrylonitrile copolymers having less than about 20mole-percent acrylonitrile, and styrene-2,2,3,3,-tetrafluoro-propylmethacrylate copolymers. Further examples are given in U.S. Pat. No.6,673,447, hereby incorporated by reference.

A hydrophilic surface region of a tubular nanostructure includes atubular nanostructure with a surface structure, e.g., a carbon surfacestructure, and/or a linker molecule having a hydrophilic portionadsorbed onto the tubular nanostructure, e.g., polyethylene glycol(PEG). The hydrophilic surface region may include one or more of amines,amides, charged or polar amino acids, alcohols, carboxylic groups,oxides, ester groups, ether groups, or ester-ether groups, ketones,aldehydes, or derivatives thereof. In one aspect, the hydrophilicsurface region includes PEG, which refers to a polymer with thestructure (—CH₂CH₂O—)_(n) that is synthesized normally by ring openingpolymerization of ethylene oxide. The PEG will impart water (and serum)solubility to the hydrophobic nanoparticle and lipid portion of thepolar lipid. The polymer is usually linear at molecular weights (MWs)less than or equal to 10 kD. The PEG will have an MW below 5,400,preferably below 2,000, or about 45 repeating ethylene oxide units.However, the higher MW PEGs (higher “n” repeating units) may have somedegree of branching. Polyethylene glycols of different MWs have alreadybeen used in pharmaceutical products for different reasons (e.g.,increase in solubility of drugs). Therefore, from the regulatorystandpoint, they are very attractive for further development as drug orprotein carriers. The PEG used here should be attached to thenanoparticles at a density adjusted for the PEG length. For example,with PL-PEG 2000, we have an estimate of 4 nm spacing between PEG chainalong the tube. At this spacing, PEG5400 is too long and starts to blockinteraction with cell surface. For PEG at approximately 1 nm distance,the PEG MW should be less than about 200, to allow hydrophobicity.

In some instances, the one or more tubular nanostructures may befunctionalized with one or more ligands, therapeutic compounds, toxin,marker, or combinations thereof. The functionalized component may be asmall chemical compound. Small chemical compounds that might be added toa tubular nanostructure include, but are not limited to, targetingbiomolecules, e.g., receptor binding ligands; therapeutic biomolecules,e.g., therapeutic small chemical compound drugs; toxins, e.g.,chemotherapy agents; and markers, e.g., fluorescent dyes and/orradioactive compounds. Any of a number of homobifunctional,heterofunctional, and/or photoreactive cross linking agents may be usedto bind biomolecules to tubular nanostructures. Examples ofhomobifunctional cross linkers include, but are not limited to, primaryamine/primary amine linkers. Examples of heterofunctional cross linkersinclude, but are not limited to, primary amine/sulfhydryl linkers.

The one or more tubular nanostructures may be further functionalizedwith ligands as therapeutic agents, including but not limited to,anti-cancer therapeutic agents, anti-microbial therapeutic agents. Theone or more tubular nanostructures may be further functionalized withmarkers to identify a cell target, e.g., a fluorescent marker, aradioactive marker, a quantum dot, a contrast agent for magneticresonance imaging (MRI) marker, a ligand reaction activated marker,lipid membrane reactive marker, cell environment reactive marker, orcombinations thereof.

A composite tubular nanostructure may comprise two or more tubularnanostructures each including a hydrophobic surface region, eachhydrophobic region flanked by two hydrophilic surface regions configuredto form a pore in a lipid bilayer membrane. For example, the compositetubular nanostructure can include 3 tubular nanostructures or 7 tubularnanostructures. Composite tubular nanostructures may be used to createmultiple pores at one or more sites in the targeted lipid bilayer.Tubular nanostructures or composite tubular nanostructures may bemodified to facilitate one or more elements to control transport ofmolecules through the tubular nanostructure. In one aspect, the one ormore elements includes at least one second ligand configured toreversibly bind a cognate of interest, wherein the cognate of interestpasses through the pore. In another aspect, the one or more elements canreversibly block the pore. Tubular nanostructures or composite tubularnanostructures may be further modified to facilitate active transport,facilitated transport, or passive transport of biomolecules through thepores formed by the nanotubes in the lipid bilayer. Active transportrequires an external energy source, e.g., the hydrolysis of ATP totransport biomolecules such as ions against a concentration gradient,the biomolecules moving, for example, from low to high concentration.

The tubular nanostructures may be modified in such a manner as to allowtransit of the nanotubes through the plasma membrane with subsequenttargeting and insertion into the lipid bilayer of one or more internalorganelles, e.g., mitochondria. Once targeted to the lipid bilayer ofthe organelle membrane, the tubular nanostructure may form pores thatenable active transport, facilitated transport, or passive transport ofcontents into or out of the organelle. In certain organelles, disruptionof the lipid bilayer may lead to cell death. In one example, tubularnanostructures may be selectively directed to the outer membrane ofmitochondria in target cells where they insert into and disrupt theouter mitochondrial membrane leading to target cell death. The tubularnanostructures having hydrophobic surface region flanked by twohydrophilic surface regions for insertion and retention in a lipidbilayer may be modified in such a manner as to mask the hydrophilic endsand allow transit through the plasma membrane. In one embodiment, thehydrophilic ends of the tubular nanostructure are modified with ahydrophobic moiety through a chemical bond that may be cleaved once thenanotube has passed into the cell.

With reference to the figures, and with reference now to FIGS. 1, 2, and3, depicted is one aspect of a system that may serve as an illustrativeenvironment of and/or for subject matter technologies, for example, atubular nanostructure which comprises a hydrophobic surface regionflanked by two hydrophilic surface regions configured to form a pore ina lipid bilayer membrane, and at least one ligand configured to bind oneor more cognates on the membrane, or for example, a tubularnanostructure which comprises a surface region configured to passthrough a lipid bilayer membrane of a cell, and a hydrophobic surfaceregion flanked by two hydrophilic surface regions configured to form apore in a lipid bilayer membrane of a cellular organelle. Accordingly,the present application first describes certain specific exemplarymethods of FIGS. 1, 2, and 3; thereafter, the present applicationillustrates certain specific exemplary methods. Those having skill inthe art will appreciate that the specific methods described herein areintended as merely illustrative of their more general counterparts.

Continuing to refer to FIG. 1, depicted is a partial diagrammatic viewof an illustrative embodiment of a tubular nanostructure or a compositetubular nanostructure and a method for inserting a tubular nanostructureor a composite tubular nanostructure into a lipid bilayer membrane. InFIG. 1A, a tubular nanostructure 100 includes a hydrophobic surfaceregion 110 flanked by two hydrophilic surface regions 120 is configuredto form a pore 170 in a lipid bilayer membrane 150, 160. The tubularnanostructure 100 further includes at least one ligand 130 configured tobind one or more cognates 140 on the lipid bilayer membrane 150, 160. InFIG. 1B, the tubular nanostructure 100 includes the at least one ligand130 configured to bind to the one or more cognates 140 on the membrane150, 160. The one or more cognates 140 may be in various positionsrelative to the extracellular side 160 of the membrane and theintracellular side 150 of the membrane. In FIG. 1C, the tubularnanostructure 100 including the hydrophobic surface region 110 flankedby two hydrophilic surface regions 120 is integrated into the lipidbilayer membrane 150, 160 of the cell. The tubular nanostructure isconfigured to form a pore 170 in the lipid bilayer membrane 150, 160. InFIG. 1D, the tubular nanostructure 100 includes the at least one ligand130 configured to bind to the one or more cognates 140 on the membrane150, 160. In this aspect, the at least one ligand 130 is configured tobind to the one or more cognates 140 on the intracellular side 150 ofthe membrane. The tubular nanostructure is configured to form a pore 170in the lipid bilayer membrane 150, 160 of the cell.

Continuing to refer to FIG. 2, depicted is a partial diagrammatic viewof an illustrative embodiment of a tubular nanostructure or a compositetubular nanostructure and a method for inserting a tubular nanostructureor a composite tubular nanostructure into a lipid bilayer membrane of acellular organelle. In FIGS. 2A and 2B, a tubular nanostructure 200which comprises a surface region 210 is configured to pass through alipid bilayer membrane 250, 260 of a cell. The lipid bilayer membrane ofthe cell has an extracellular side 260 of the membrane and anintracellular side 250 of the membrane In FIG. 2C, the tubularnanostructure further includes a hydrophobic surface region 220 flankedby two hydrophilic surface regions 225 configured to form a pore 290 ina lipid bilayer membrane 270, 280 of a cellular organelle. The tubularnanostructure 200 which comprises a surface region 210 is configured topass through a lipid bilayer membrane 250, 260 of the cell. The tubularnanostructure 200 is configured to interact with a cellular component285 to produce the at least one tubular nanostructure including thehydrophobic surface region 220 flanked by two hydrophilic surfaceregions 225. The tubular nanostructure 200 may further include at leastone ligand 230 configured to bind one or more cognates 240 on the lipidbilayer membrane 270, 280 of the cellular organelle. The one or morecognates 240 may be in various positions relative to the cytoplasmicside 280 of the lipid bilayer membrane or the intraorganellar side 270of the lipid bilayer membrane of the cellular organelle. In FIG. 2D, thetubular nanostructure 200 including the hydrophobic surface region 220flanked by two hydrophilic surface regions 225 is integrated into thelipid bilayer membrane 270, 280 of the cellular organelle. The tubularnanostructure is configured to form a pore 290 in the lipid bilayermembrane 270, 280. In FIG. 2E, the tubular nanostructure 200 may includethe at least one ligand 230 configured to bind to the one or morecognates 240 on the membrane 270, 280 of a cellular organelle. In thisaspect, the at least one ligand 230 is configured to bind to the one ormore cognates 240 on the intraorganellar side 270 of the membrane. Thetubular nanostructure is configured to form a pore 290 in the lipidbilayer membrane 270, 280 of the cellular organelle.

FIG. 3 depicts some exemplary aspects of a method as that described inFIGS. 1 and 2. FIG. 3 illustrates an exemplary method 300 for insertinga tubular nanostructure into a lipid bilayer membrane. The methodincludes applying 302 to a lipid bilayer membrane, a tubularnanostructure including a hydrophobic surface region flanked by twohydrophilic surface regions configured to form a pore in the lipidbilayer membrane and including at least one ligand configured to bindone or more cognates on the membrane, under conditions and for timesufficient to allow the nanostructure to penetrate the membrane

FIG. 4 illustrates an exemplary method 400 for providing a stable porein a lipid bilayer membrane. The method includes positioning 402 acrossa lipid bilayer membrane a tubular nanostructure including a hydrophobicsurface region flanked by two hydrophilic surface regions configured toform a pore in the lipid bilayer membrane and including at least oneligand configured to bind one or more cognates on the membrane.

FIG. 5 illustrates an exemplary method 500 for inserting a tubularnanostructure into a lipid bilayer membrane. The method includesapplying 502 to a lipid bilayer membrane a composite tubularnanostructure including two or more nanotubes wherein at least onenanotube includes a hydrophobic surface region flanked by twohydrophilic surface regions configured to form a pore in a lipid bilayermembrane, under conditions and for time sufficient to allow thecomposite nanostructure to penetrate the membrane.

FIG. 6 illustrates an exemplary method 600 for providing a pore in alipid bilayer membrane. The method includes positioning 602 across alipid bilayer membrane a composite tubular nanostructure including twoor more nanotubes wherein at least one nanotube includes a hydrophobicsurface region flanked by two hydrophilic surface regions configured toform a pore in a lipid bilayer membrane.

FIG. 7 illustrates an exemplary method 700 for disrupting a lipidbilayer membrane of a cell. The method includes contacting 702 the cellwith at least one tubular nanostructure including a hydrophobic surfaceregion flanked by two hydrophilic surface regions configured to form apore in the lipid bilayer membrane and including at least one ligandconfigured to bind one or more cognates on the lipid bilayer membrane ofthe cell.

Tubular Nanostructure

Tubular nanostructures as described herein may be made from a widevariety of materials, for example, organic, inorganic, polymeric,biodegradable, biocompatible and combinations thereof. Non-limitingexamples of inorganic materials to make tubular nanostructures asdescribed herein include iron oxide, silicon oxide, titanium oxide andthe like. Examples of biodegradable monomers formed into tubularnanostructures include polysaccharides, cellulose, chitosan,carboxymethylated cellulose, polyamino-acids, polylactides andpolyglycolides and their copolymers, copolymers of lactides andlactones, polypeptides, poly-(ortho)esters, polydioxanone,poly-β-aminoketones, polyphosphazenes, polyanhydrides,polyalkyl(cyano)acrylates, poly(trimethylene carbonate) and copolymers,poly(ε-caprolactone) homopolymers and copolymers, polyhydroxybutyrateand polyhydroxyvalerate, poly(ester)urethanes and copolymers,polymethyl-methacrylate and combinations thereof. The carrier may eveninclude or made from polyglutamic or polyaspartic acid derivatives andtheir copolymers with other amino-acids.

The tubular nanostructure as described herein may be a carbon nanotube.Carbon nanotubes are all-carbon hollow graphitic tubes with nanoscalediameter. They can be classified by structure into two main types:single walled CNTs (SWNTs), which consist of a single layer of graphenesheet seamlessly rolled into a cylindrical tube, and multiwalled CNTs(MWNTs), which consist of multiple layers of concentric cylinders.Carbon sources for use in generating carbon nanotubes include, but arenot limited to, carbon monoxide and hydrocarbons, including aromatichydrocarbons, e.g., benzene, toluene, xylene, cumene, ethylbenzene,naphthalene, phenanthrene, anthracene or mixtures thereof, non-aromichydrocarbons, e.g., methane, ethane, propane, ethylene, propylene,acetylene or mixtures thereof; and oxygen-containing hydrocarbons, e.g.,formaldehyde, acetaldehyde, acetone, methanol, ethanol or mixturesthereof.

Carbon nanotubes may be synthesized from one or more carbon sourcesusing a variety of methods, e.g., arc-discharge, laser ablation, orchemical vapor deposition (CVD; see, e.g., Bianco, et al., inNanomaterials for Medical Diagnosis and Therapy. pp. 85-142.Nanotechnologies for the Live Sciences Vol. 10 Edited by Challa S. S. R.Kumar, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007, which isincorporated herein by reference).

Carbon nanotubes may be synthesized using the arc discharge method whichcreates nanotubes through arc-vaporization of two carbon rods placed endto end, separated by approximately 1 mm, in an enclosure that is filled,for example, with inert gas (e.g., helium, argon) at low pressure(between 50 and 700 mbar). A direct current of 50 to 100 amperes drivenby approximately 20 volts creates a high temperature discharge betweenthe two electrodes. The discharge vaporizes one of the carbon rods andforms a small rod shaped deposit on the other rod.

Alternatively, carbon nanotubes may be synthesized using laser ablationin which a pulsed or continuous laser energy source is used to vaporizea graphite target in an oven at 1200° C. The oven is filled with aninert gas such as helium or argon, for example, in order to keep thepressure at 500 Torr. A hot vapor plume forms, expands, and coolsrapidly. As the vaporized species cool, small carbon molecules and atomsquickly condense to form larger clusters. The catalysts also begin tocondense and attach to carbon clusters from which the tubular moleculesgrow into single-wall carbon nanotubes. The single-walled carbonnanotubes formed in this case are bundled together by van der Waalsforces.

Carbon nanotubes may also be synthesized using chemical vapor deposition(CVD). CVD synthesis is achieved by applying energy to a gas phasecarbon source such as methane or carbon monoxide, for example. Theenergy source is used to “crack” the gas molecules into reactive atomiccarbon. The atomic carbon diffuses towards a substrate, which is heatedand coated with a catalyst, e.g., Ni, Fe or Co where it will bind. Thecatalyst is generally prepared by sputtering one or more transitionmetals onto a substrate and then using either chemical etching orthermal annealing to induce catalyst particle nucleation. Thermalannealing results in cluster formation on the substrate, from which thenanotubes will grow. Ammonia may be used as the etchant. Thetemperatures for the synthesis of nanotubes by CVD are generally withinthe 650-900° C. range. A number of different CVD techniques forsynthesis of carbon nanotubes have been developed, such as plasmaenhanced CVD, thermal chemical CVD, alcohol catalytic CVD, vapor phasegrowth, aero gel-supported CVD and laser-assisted thermal CVD, and highpressure CO disproportionation process (HiPCO). Additional methodsdescribing the synthesis of carbon nanotubes may be found, for example,in U.S. Pat. Nos. 5,227,038; 5,482,601; 6,692,717; 7,354,881 which areincorporated herein by reference.

Carbon nanotubes may be synthesized as closed at one or both ends. Assuch, forming a hollow tube may necessitate cutting the carbonnanotubes. Carbon nanotubes may be cut into smaller fragments using avariety of methods including but not limited to irradiation with highmass ions, intentional introduction of defects into the carbon nanotubeduring synthesis, sonication in the presence of liquid or moltenhydrocarbon, lithography, oxidative etching with strong oxidatingagents, mechanical grinding with diamond balls, or physical cutting withan ultra microtome (see, e.g., U.S. Pat. No. 7,008,604; Wang et al,Nanotechnol. 18:055301, 2007, which are incorporated herein byreference). For irradiation with high mass ions, for example, the carbonnanotubes are subjected to a fast ion beam, e.g., from a cyclotron, atenergies of from about 0.1 to 10 giga-electron volts. Suitable high massions include those over about 150 AMU's such as bismuth, gold, uraniumand the like. To generate defects that are susceptible to cleavage, thecarbon nanotubes may be synthesized in the presence of a small amount ofboron, for example. For sonication, carbon nanotubes may be sonicated inthe presence of 1,2-dichloroethane, for example, using a sonicator withsufficient acoustic energy over a period ranging from 10 minutes to 24hours, for example. For oxidative etching, carbon nanotubes may beincubated in a solution containing 3:1 concentrated sulfuric acid:nitricacid for 1 to 2 days at 70° C. For cutting with an ultra microtome, thecarbon nanotubes are magnetically aligned, frozen to a temperature ofabout −60° C., and cut using an ultra-thin cryo-diamond knife.

Once synthesized, carbon nanotubes may be further purified to eliminatecontaminating impurities, e.g., amorphous carbon and catalyst particles.Methods for further purification include, but are not limited to, acidoxidation, microfiltration, chromatographic procedures, microwaveirradiation, and polymer-assisted purification (see, e.g., U.S. Pat. No.7,357,906, which is incorporated herein by reference). Chromotagraphyand microfiltration may also be used to isolate a uniformed populationof carbon nanotubes with similar size and diameter, for example (see,e.g., Bianco, et al., in Nanomaterials for Medical Diagnosis andTherapy. pp. 85-142. Nanotechnologies for the Live Sciences Vol. 10Edited by Challa S. S. R. Kumar, WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim, 2007, which is incorporated herein by reference).Alternatively, purified carbon nanotubes may be purchased from acommercial source (from, e.g., Carbon Nanotechologies, Houston, Tex.;Sigma-Aldrich, St. Louis, Mo.).

Alternatively, a tubular nanostructure as described herein may be apeptide nanotube. Peptide nanotubes are extended tubular beta-sheet-likestructures and are constructed by the self-assembly of flat, ring-shapedpeptide subunits made up of alternating D- and L-amino acid residues asdescribed in U.S. Pat. Nos. 6,613,875 and 7,288,623, and in Hartgerink,et al., J. Am. Chem. Soc. 118:43-50, 1996, which are incorporated hereinby reference. For example, gramicidin is a pentadecapeptide which formsa β-helix with a hydrophilic interior and a lipophilic exterior bearingamino acid side chains in membranes and nonpolar solvents. In thisinstance, the helix length is approximately half of the thickness of alipid bilayer and as such, two gramicidin molecules form an end-to-enddimer stabilized by hydrogen bonds that spans the lipid bilayer. Peptidenanotubes are constructed by highly convergent noncovalent processes bywhich cyclic peptides rapidly self-assemble and organize into ultralarge, well ordered three-dimensional structures, upon an appropriatechemical- or medium-induced triggering. The properties of the outersurface and the internal diameter of peptide nanotubes may be adjustedby the choice of the amino acid side chain functionalities and the ringsize of the peptide subunit employed.

Alternatively, a tubular nanostructure as described herein may be alipid nanotube. Lipid nanotubes are typically formed usingself-assembling microtubule-forming diacetylenic lipids, such as complexchiral phosphatidylcholines, and mixtures of these diacetylenic lipidsas described in U.S. Pat. Nos. 4,877,501, 4,911,981 and 4,990,291, whichare incorporated herein by reference. The synthesis of self-assemblinglipid nanotubes may be accomplished by combining the appropriate lipidswith an alcohol and a water phase which leads to the production of lipidmicrocylinders by direct crystallization. The formation of the lipidtubules may be modulated by the choice of alcohol and/or combination ofalcohols, the ratio of alcohol to water, and variations in the reactiontemperature (see, e.g., U.S. Pat. No. 6,013,206, which is incorporatedherein by reference). A simple method for generating uniform lipidnanotubes from single-chain diacetylene secondary amine salts has beendescribed in Lee, et al., J. Am. Chem. Soc. 126:13400-13405, 2004, whichis incorporated herein by reference.

Functionalization of Tubular Nanostuctures for Targeting and Insertioninto a Cellular Membrane

Tubular nanostructures as described herein may be functionalized toinclude hydrophilic surface regions at one or both ends of the tubularnanostructure to facilitate insertion and retention of the tubularnanostructure into a lipid bilayer membrane associated with a targetcell or organelle (see, e.g., U.S. Patent Application 2004/0023372 A1,which is incorporated herein by reference). The hydrophilic surfaceregion may include one or more of amines, amides, charged or polar aminoacids, alcohols, carboxylic groups, oxides, ester groups, ether groups,or ester-ether groups, ketones, aldehydes, or derivatives thereof.Tubular nanostructures may be further functionalized to include one ormore ligand, one or more therapeutic compounds, one or more toxins, oneor more markers, or combinations thereof. A tubular nanostructure may befunctionalized using non-covalent and covalent methodologies.

Non-covalent functionalization of carbon nanotubes, for example, may beaccomplished using π-π stacking interactions between conjugatedmolecules and the graphitic sidewall of the tubular nanostructure. Forexample, compounds with a pyrene moiety, e.g.,N-succinimidyl-1-pyrenebutanoate may be irreversibly absorbed onto thesurface of a carbon nanotube through π-π stacking interaction. In thisinstance, the succinimidyl ester group associated with thepyrenebutonaote may be used to link to primary or secondary amines andas such may be used to couple biomolecules, e.g., proteins and nucleicacids to the tubular nanostructure. Other molecules that may be linkedto a tubular nanostructure via π-π stacking interactions include thephotosensitizers phthalocyanines and porphyrins (see, e.g., Bianco, etal., in Nanomaterials for Medical Diagnosis and Therapy. pp. 85-142.Nanotechnologies for the Live Sciences Vol. 10 Edited by Challa S. S. R.Kumar, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007, which isincorporated herein by reference).

Alternatively, non-covalent functionalization may be accomplished usinghydrophobic interactions with amphiphilic molecules. In this instance,the hydrophobic surface of the amphiphilic molecules interactnoncovalently with the aromatic surface of the carbon nanotube whileexposing their hydrophilic parts to the aqueous medium, allowing forsolubilization of hydrophobic tubular nanostructures in aqueoussolutions. Examples of molecules that may be used for this purposeinclude, but are not limited to, water-soluble polymers, e.g.,polyvinylpyrrolidone and polystyrenesulfonate; surfactants, e.g.,anionic, nonionic, and cationic surfactants including, for example,deoxycholic acid, taurodeoxycholic acid, sodium dodecylbenzenesulfonate, and sodium dodecyl sulfate; amphiphilic peptides, and singlestranded DNA. In addition, a biomolecule may be attached indirectly to atubular nanotube, e.g., a carbon nanotube through an amphiphilicbifunctional linker, e.g., phospholipid (PL)-poly(ethylene glycol) (PEG)chains and terminal amine (PL-PEG-NH₂) in which the PL alkyl chainsinteract noncovalently with the carbon nanotube and the amine group maybe used to link to biomolecules. Other examples of functionalized PEGlipids include, but are not limited to, phospholipid-PEG-carboxylicacid, phospholipid-PEG-maleimide, and phospholipid-PEG-biotin, forexample. For example, the phospholipid-PEG-biotin derivative1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol]2000] (DSPE-PEG(2000)-biotin) may be added to carbon nanotubes bysonication, followed by centrifugation to isolate the functionalizednanotubes (see, e.g., Chakravarty, et al., Proc. Natl. Acad. Sci. USA105:8697-8702, 2008, which is incorporated herein by reference).Similarly, DNA or RNA may be linked to a carbon nanotube using aheterofunctional crosslinker, e.g., sulfosuccinimidyl6-(3′-[2-pyridyldithio]propionamido)hexanoate (sulfo-LC-SPDP). (see,e.g., Bianco, et al., in Nanomaterials for Medical Diagnosis andTherapy. pp. 85-142. Nanotechnologies for the Live Sciences Vol. 10Edited by Challa S. S. R. Kumar, WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim, 2007, which is incorporated herein by reference).

Tubular nanostructures may also be functionalized using covalentinteractions. Covalent functionalization of carbon nanotubes, forexample, may involve defect functionalization and/or side-wallfunctionalization. Defect functionalization takes advantage of defectsin the carbon nanotube structure characterized by disruptions in thesix-membered rings of the graphene sheets such as might be found at thecut ends of carbon nanotubes. Defect functionalization may also bepresent on the side-walls, characterized by the presence of five- andseven-membered rings within the graphene sheet of six-membered rings.Treatment of carbon nanotubes with strong oxidizing agents, e.g., nitricacid, KMnO₄/H₂SO₄, O₂, K₂Cr₂O₇/H₂SO₄ or OsO₄ may be used to cut carbonnanotubes, generating open ends and creating a hollow tube (see, e.g.,U.S. Pat. No. 7,008,604, which is incorporated herein by reference).Oxidation may also be used to add functional groups, e.g., carboxylicacid, ketone, alcohol and ester groups to the ends and defect sites onthe side-walls and as such may be used to create hydrophilic surfaceregions.

The functional groups added to carbon nanotubes by oxidation, forexample, may be used to further modify the ends and/or the side walls ofthe nanotubes. For example, carboxylic acid moieties on the nanotube maybe used to form amide and ester linkages. In this instance, reactiveintermediates are formed by treating the carboxylic acid groups withthionyl chloride, carbodiimide, or N-hydroxysuccinimide (NHS). Thereactive intermediates are then able to form covalent linkages withbiomolecules, e.g., polymers suchpoly-propionyl-ethylenimine-co-ethylenimine (PPEI-EI),poly-n-vinylcarbazole (PVK-PS) and polyethylene glycol (PEG),poly-n-butyl methacrylate (PnBMA), poly-methyl methacrylate (PMMA), andPMMA-b-poly-hydroxyethyl methacrylate (PHEMA); proteins such as bovineserum albumin; DNA molecules; and other biomolecules, e.g., biotin.

End and/or side-wall functionalization of a tubular nanostructure may beaccomplished using various chemical reactions including but not limitedto fluorination, radical addition, nucleophilic addition, electrophilicaddition, and cycloaddition, for example (see, e.g., Bianco, et al., inNanomaterials for Medical Diagnosis and Therapy. pp. 85-142.Nanotechnologies for the Live Sciences Vol. 10 Edited by Challa S. S. R.Kumar, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007, which isincorporated herein by reference). Fluorine may be added to the surfaceof a carbon nanotube, for example, by heating the nanotube in thepresence of elemental fluorine at temperatures ranging from 150 to 600°C. (see, e.g., U.S. Pat. No. 6,841,139, which is incorporated herein byreference). The fluorine group on the carbon nanotube may be furthersubstituted with strong nucleophilic reagents, e.g., Grignard,alkyllithium reagents and/or metal alkoxides. Alternatively, a tubularnanostructure, e.g., a carbon nanotube, may be functionalized bycycloaddition with, for example, dichlorocarbene, nitrenes,bromomalonates, o-quinodimethane, azido group, alkyne/azide, and/orazomethine ylides. For example, protected amino groups may be introducedonto the surface of carbon nanotubes using 1,3-dipolar cycloaddition ofazomethine ylides. The N-protected amino acid may then be used to linkbiomolecules, e.g., bioactive peptides (see, e.g., Pantorotto, et al.,J. Am. Chem. Soc. 125:6160-6164, 2003, which is incorporated herein byreference).

In some instances, it may be beneficial to selectively functionalize oneportion or portions of the ends and/or sidewalls of a tubularnanostructure. Asymmetric functionalization of carbon nanotubes may beaccomplished using a masking technique. For example, carbon nanotubesmay be partially embedded in a polymer matrix, including, but notlimited to, poly(dimethylsiloxane), polystyrene, poly(methylmethacrylate), or polydiene rubber or a combination thereof and thenon-embedded or exposed portion functionalized (see, e.g., Qu & Dai,Chem. Commun. 37: 3829-3861, 2007, which is incorporated herein byreference). An organic solvent, e.g., toluene, may be used to wash awaythe masking polymer. Asymmetric functionalization of the ends of carbonnanotubes may be accomplished using a lithographic procedure to cut thenanotubes followed by chemical modification of the exposed tube ends viaplasma treatment while the tube side-walls remain protected by a resistlayer (see, e.g., Burghard Small 1:1148-1140, 2005, which isincorporated herein by reference).

Alternatively, asymmetric functionalization of carbon nanotubes may beaccomplished by floating the nanotubes on a photoreactive solution withonly one side of the nanotube in contact with the solution and exposingthe solution to UV light (see, e.g., U.S. Patent Application2006/0257556 A1, which is incorporated herein by reference).Photoreactive reagents are chemically inert reagents that becomereactive when exposed to ultraviolet or visible light and areexemplified by derivatives of aryl azides. When an aryl azide is exposedto UV light, it forms a nitrene group that can initiate additionreactions with double bonds, insertion into C-H and N-H sites, orsubsequent ring expansion to react with a nucleophile (e.g., primaryamines). Examples of photoreactive cross linkers include, but are notlimited, to primary amine linkers such as ANB-NOS(N-5-azido-2-nitrobenzyloxysuccinimide), NHS-ASA(N-hydroxy-succinimideyl-4-azidosalicyclic acid), Sulfo HSAB(N-hydroxysulfosuccinimidyl-4-azidobenzoate), Sulfo SAED(sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3-dithiopropionate),Sulfo SAND (sulfosuccinimidyl2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-propionate), Sulfo SANPAH(sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino) hexanoate), SulfoSADP (sulfosuccinimidyl (4-azidophenyldithio) propionate, and Sulfo SASD(sulfosuccinimidyl-2-(rho-azidosalicylamido)ethyl-1,3-dithiopropionate;carbohydrate linkers such as ABH (azidobenzoyl hydrazide); argininelinkers such as APG (azidophenyl glyoxal monohydrate), sulfhydryllinkers such as APDP (N-(4[rho-azidosalicylamido]butyl)-3′-(2′-pyridyldithio) propionamide); non selective linkers suchas BASED (bis(beta-[4-azidosalicylamido]-ethyl) disulfide).

Tubular nanostructures may be functionalized to include one or moreligand, therapeutic compound, toxin, marker, or combinations thereof. Insome instances the one or more ligand, therapeutic compound, toxinand/or marker is a protein biomolecule. Protein biomolecules that mightbe added to a tubular nanostructure include, but are not limited to,targeting biomolecules, e.g., antibodies, receptor ligands, and lectins;therapeutic biomolecules, e.g., therapeutic proteins or peptides;transporter biomolecules, e.g., components of the ATP-binding cassette(ABC) transporters; pore-forming agents such as antimicrobial peptides;and toxic biomolecules such as protein-based plant and bacterial toxins.The tubular nanostructure may be functionalized with amines, carboxylicacids, thiols, aldehydes and combinations thereof to facilitate linkageto protein biomolecules. For example, attachment of one or more proteinmolecules to a carbon nanotube may be performed using heterobifunctionalcrosslinkers. For example, a heterobifunctional crosslinker may be addedcovalently to a carbon nanotube by adding amino groups to the nanotubevia azomethine ylide cycloaddition or alkyne azide cycloaddition,followed by derivatization of the amino groups with a heterobifunctionalcrosslinker, e.g.,succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)(LC-SMCC). The nanotube functionalized in this manner is combined with aprotein into which reactive sulfhydryl groups have been introduced with2-iminothiolane-HCl (see, e.g., McDevitt, et al., J. Nucl. Med.48:1180-1189, 2007, which is incorporated herein by reference).Alternatively, a protein biomolecule may be added to a tubularnanostructure such as a carbon nanotube, for example, by non-covalentattachment of phospholipid-PEG-NH₂ to the nanotube and covalentinteraction of the associated amine group with thiolated protein (see,e.g., Welsher, et al., Nano Lett. 8:586-590, 2008, which is incorporatedherein by reference). Alternatively, a protein biomolecule may be addedto a tubular nanostructure using a biotin/avidin linkage in which thecarbon nanotubes are functionalized with biotin using aphospholipid-PEG-biotin as described herein and combined with avidin- orstreptavidin-modified protein. A protein may be modified with avidin,for example, by activating the avidin withm-maleimidobenzoyl-N-hydroxysuccinimide ester and linking it tothiolated target protein (see, e,g, Chakravarty, et al., Proc. Natl.Acad. Sci. USA, 105: 8697-8702, 2008, which is incorporated herein byreference).

Biomolecules such as antibodies, for example, may also be attached topeptide nanotubes and boron nitride nanotubes (see, e.g., Zhao & MatsuiSmall 3:1390-1393, 2007; U.S. Patent Application 2006/0067941, which areincorporated herein by reference). For example, boron nitride nanotubesmay be chemically modified with primary amines such as methylamine andethanolamine that may be used for additional functionalization of thenanotubes (Wu, et al., J. Am. Chem. Soc. 128:12001-12006, 2006, which isincorporated herein by reference).

One or more tubular nanostructures may be functionalized with one ormore peptides. In some instances, one or more peptides may be linked toa tubular nanostructure using the methods described above for proteins.Alternatively, one or more peptides may be linked to a tubularnanostructure using fragment condensation of fully protected peptidesand/or selective chemical ligation (see, e.g., U.S. Patent Application20060199770; Pantarotto, et al., J. Am. Chem. Soc. 125:6160-6164, 2003,which are incorporated herein by reference). For selective chemicalligation, for example, carbon nanotubes may be functionalized withprimary amines and N-succinimidyl 3-maleimidopropionate and reacted withN-terminal acetylated peptide to form peptide-carbon nanotubeconjugates. Alternatively, peptides may be designed using phage displaymethodologies that selectively recognize and bind carbon nanotubes asdescribed in U.S. Pat. No. 7,304,128, which is incorporated herein byreference.

In some instances, the one or more tubular nanostructures may befunctionalized with one or more ligand, therapeutic compound, toxin,marker, or combination thereof that is a polynucleotide biomolecule.Polynucleotide biomolecules that might be added to a tubularnanostructure include, but are not limited to, aptamers, antisense RNA,RNAi, DNA, or combinations thereof. For example, DNA may be added to atubular nanostructure such as a carbon nanotube using astreptavidin-biotin linkage. In this instance, streptavidin may benon-covalently associated with the carbon nanotube and combined withbiotin modified DNA. Alternatively, single strand DNA may be bound to acarbon nanotube by direct non-covalent interaction forming a coil aroundthe nanotube. Alternatively, a small oligonucleotide such as an aptamer,for example, may be linked to a carbon nanotube using carbodiimidazole(CDI)-Tween (see, e.g., So, et al., J. Am. Chem. Soc. 127:11906-11907,2005, which is incorporated herein by reference). Alternatively, a DNAor RNA aptamer may be linked to a carbon nanotube via astreptavidin-biotin linkage. In this instance, biotin may be introducedinto the DNA or RNA aptamer during synthesis of the aptamer and thenbound to streptavidin associated with the carbon nanotube.Alternatively, a DNA or RNA aptamer may be conjugated to a tubularnanotube using amine- or sulfhydryl-reactive crosslinkers (e.g., fromPierce-Thermo Scientific, Rockford, Ill., USA) using the methodsdescribed herein. As such, the aptamer may be synthesized in thepresence of specific bases modified with primary amines or thiols.

In some instances, the one or more tubular nanostructures may befunctionalized with one or more ligand, therapeutic compound, toxin,marker, or combinations thereof as a small chemical compound. Smallchemical compounds that might be added to a tubular nanostructureinclude, but are not limited to, targeting biomolecules, e.g., receptorbinding ligands; therapeutic biomolecules, e.g., therapeutic smallchemical compound drugs; toxins, e.g., chemotherapy agents; and markers,e.g., fluorescent dyes and/or radioactive compounds. For example,reversible attachment of a platinum based chemotherapy to a carbonnanotube can be used in which the platinum compound was modified with alinker arm and an N-succinimidyl ester group which readily formed amidelinkages with PEG-tethered primary amines on the surface of carbonnanotubes (Feazell, et al., J. Am. Chem. Soc. 129:8438-8439, 2007, whichis incorporated herein by reference).

In general, any of a number of homobifunctional, heterofunctional,and/or photoreactive cross linking agents may be used to bindbiomolecules to tubular nanostructures. Examples of homobifunctionalcross linkers include, but are not limited to, primary amine/primaryamine linkers such as BSOCES ((bis(2-[succinimidooxy-carbonyloxy]ethyl)sulfone), DMS (dimethyl suberimidate), DMP (dimethyl pimelimidate), DMA(dimethyl adipimidate), DSS (disuccinimidyl suberate), DST(disuccinimidyl tartate), Sulfo DST (sulfodisuccinimidyl tartate), DSP(dithiobis(succinimidyl propionate), DTSSP (3,3′-dithiobis(succinimidylpropionate), EGS (ethylene glycol bis(succinimidyl succinate)) andsulfhydryl/sulfhydryl linkers such as DPDPB(1,4-di-(3′-[2′pyridyldithio]-propionamido) butane). Examples ofheterofunctional cross linkers include, but are not limited to, primaryamine/sulfhydryl linkers such as MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo MBS(m-maleimidobenzoyl-N-hydroxysulfosuccinimide), GMBS(N-gamma-maleimidobutyryl-oxysuccinimide ester), Sulfo GMBS(N-gamma-maleimidobutyryloxysulfosuccinimide ester), EMCS(N-(epsilon-maleimidocaproyloxy) succinimide ester), Sulfo EMCS(N-(epsilon-maleimidocaproyloxy) sulfo succinimide), STAB(N-succinimidyl(4-iodoacetyl)aminobenzoate), SMCC (succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SMPB (succinimidyl4-(rho-maleimidophenyl) butyrate), Sulfo STAB(N-sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), Sulfo SMCC(sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate),Sulfo SMPB (sulfosuccinimidyl 4-(rho-maleimidophenyl) butyrate), andMAL-PEG-NHS (maleimide PEG N-hydroxysuccinimide ester);sulfhydryl/hydroxyl linkers such as PMPI (N-rho-maleimidophenyl)isocyanate; sulfhydryl/carbohydrate linkers such as EMCH(N-(epsilon-maleimidocaproic acid) hydrazide); and amine/carboxyllinkers such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride).

Ligands Targeted to Cognates which are Associated with Target Cellsand/or Organelles

The tubular nanostructures as described herein may include one or moreligands that are configured to bind to one or more cognates associatedwith the lipid bilayer membrane of a target cell or organelle. A targetcell may include a tumor cell and/or other diseased cell type in amammalian subject. A target cell may also include a pathogen, e.g.,bacteria, fungi, and/or parasites. In some instances, the tubularnanostructures may be designed to target a specific cellular organelle,e.g., the mitochondria. One or more cognates associated with a targetcell or organelle may include at least one of a protein, a carbohydrate,a glycoprotein, a glycolipid, a sphingolipid, a glycerolipid, ormetabolites thereof.

Tumor Markers

One or more tubular nanostructures may include one or more ligands thatbind one or more cognates associated with a tumor cell. In thisinstance, the cognate may be a cell surface receptor or cell surfacemarker on a tumor cell. Examples of cognates associated with tumor cellsmay include, but are not limited to, BLyS receptor, carcinoembryonicantigen (CA-125), CD25, CD34, CD33 and CD123 (acute myeloid leukemia),CD20 (chronic lymphocytic leukemia), CD19 and CD22 (acute lymphoblasticleukemia), CD30, CD40, CD70, CD133, 57 kD cytokeratin, epithelialspecific antigen, extracellular matrix glycoprotein tenascin, Fas/CD95,gastrin-releasing peptide-like receptors, hepatocyte specific antigen,human gastric mucin, human milk fat globule, lymphatic endothelial cellmarker, matrix metalloproteinase 9, melan A, melanoma marker,mesothelin, mucin glycoproteins (e.g., MUC1, MUC2, MUC4, MUCSAC, MUC6),prostate specific antigen, prostatic acid phosphatase, PTEN, renal cellcarcinoma marker, RGD-peptide binding integrins, sialyl Lewis A,six-transmembrane epithelial antigen of the prostate (STEAP), TNFreceptor, TRAIL receptor, tyrosinase, villin. Other tumor associatedantigens include, but are not limited to, alpha fetoprotein,apolipoprotein D, clusterin, chromogranin A, myeloperoxidase, MyoD1myoglobin placental alkaline phosphatase c-fos, homeobox genes,aberrantly glycosylated antigens.

Bacterial Cognates

One or more tubular nanostructures may include one or more ligands thatbind one or more cognates associated with bacteria. A cognate onbacteria may be a component of the bacterial outer membrane, cell wall,and/or cytoplasmic membrane, for example. Examples of cognatesassociated with the bacterial outer membrane of Gram-negative bacteriainclude, but are not limited to, lipopolysaccaride and OMP (outermembrane protein) porins, the latter of which are exemplified by OmpC,OmpF and PhoP of E. coli. Examples of cognates associated with thebacterial cell wall of both Gram-positive and Gram-negative bacterialinclude, but are not limited to, peptidoglycans polymers composed of analternating sequence of N-acetylglucoamine and N-acetyl-muraminic acidand crosslinked by amino acids and amino acid derivatives. Examples ofcognates associated with the bacterial cytoplasmic membrane include, butare not limited to, the MPA1-C (also called polysaccharide copolymerase,PCP2a) family of proteins, the MPA2 family of proteins, and the ABCbacteriocin exporter accessory protein (BEA) family of proteins. Otherexamples of cognates associated with bacteria include, but are notlimited to, transporters, e.g., sugar porter (major facilitatorsuperfamily), amino-acid/polyamine/organocation (APC) superfamily,cation diffusion facilitator, resistance-nodulation-division typetransporter, SecDF, calcium:cation antiporter, inorganic phosphatetransporter, monovalent cation:proton antiporter-1, monovalentcation:proton antiporter-2, potassium transporter, nucleobase:cationsymporter-2, formate-nitrite transporter, divalent anion:sodiumsymporter, ammonium transporter, and multi-antimicrobial extrusion;channels, e.g., major intrinsic protein, chloride channel, and metal iontransporter; and primary active transporters, e.g., P-type ATPase,arsenite-antimonite efflux, Type II secretory pathway (SecY), andsodium-transporting carboxylic acid decarboxylase. A number of otherpotential cognates associated with bacteria have been described inChung, et al., J. Bacteriology 183: 1012-1021, 2001, which isincorporated herein by reference.

Mitochondrial Cognates

One or more tubular nanostructures may include one or more ligands thatbind one or more cognates associated with an organelle, e.g.,mitochondria within a tumor cell and/or other targeted cell. Examples ofcognates associated with the mitochondrial outer membrane include, butare not limited to, carnitine palmitoyl transferase 2, translocase ofouter membrane (TOM70), sorting/assembly machinery, ANT, voltagedependent anion channel (VDAC/Porin), and monoamine oxidase. In someinstances, one or more tubular nanostructures as described herein mayinclude one or more ligands that bind to one or more cognates on theinner mitochondrial membrane. A cognate of the inner mitochondrialmembrane may be a membrane associated receptor or protein, e.g., one ormore proteins associated with the carnitine acyltransferase IItransporter, NADH dehydrogenase complex (Complex I), succinatedehydrogenase (Complex II), cytochrome bc1 complex (Complex III),cytochrome c oxidase complex (Complex IV), ATP synthase, or uncouplingprotein (UCP).

Functionalization of Tubular Nanotubes with Various Ligands that Bind toCognates

A tubular nanostructure as described herein may include one or moreligands that bind one or more cognates on a target cell or organelle. Aligand that binds a cognate may include, but is not limited to, at leasta portion of an antibody, antibody-coated liposome, polynucleotide,polypeptide, receptor, viral plasmid, polymer, protein, carbohydrate,lipid, pore-forming toxin, lectin, or any combination thereof. As such,the tubular nanostructure containing one or more ligands may beselectively directed towards target cells expressing the correspondingone or more cognates. In one aspect, a protein cognate may bind to acompound having a lipid or carbohydrate moiety, e.g., a saccharide, aglycoprotein or a lipoprotein/proteolipid. The one or more ligands maybe attached to the side-walls of the tubular nanostructure.Alternatively, one or more ligands may be attached to either and/or bothends of the tubular nanostructure. Increased tissue or cell specificitymay be garnered by multifunctionalization of the tubular nanostructurewith two or more ligands directed towards two or more distinct cognateson the target tissue. In the instance where the end targets aremitochondria in specific cells, the tubular nanostructure may bemultifunctionalized, e.g., with a first ligand directed to a firstcognate on the cell membrane of the target cell and with a second liganddirected to a second cognate on the membrane of the mitochondria.

Antibody Ligands

In some instances, tubular nanostructures may be modified with one ormore ligands that are antibodies. Antibodies or fragments thereof foruse in functionalizing a tubular nanostucture may include, but are notlimited to, monoclonal antibodies, polyclonal antibodies, Fab fragmentsof monoclonal antibodies, Fab fragments of polyclonal antibodies, Fab₂fragments of monoclonal antibodies, and Fab₂ fragments of polyclonalantibodies, among others. Single chain or multiple chainantigen-recognition sites can be used. Multiple chainantigen-recognition sites can be fused or unfused. Antibodies orfragments thereof may be generated using standard methods as describedby Harlow & Lane (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press; 1^(st) edition 1988), which is incorporated herein byreference). In another embodiment, the functional group is anantigen-binding moiety, e.g., a moiety comprising theantigen-recognition site of an antibody. Alternatively, an antibody orfragment thereof directed against a cognate may be generated using phagedisplay technology (see, e.g., Kupper, et al. BMC Biotechnology 5:4,2005, which is incorporated herein by reference). A single chainantibody, for example, may also incorporate streptavidin as part of afusion protein to facilitate attachment of the antibody to the tubularnanostructure via a biotin-streptavidin linkage, for example (see, e.g.,Koo, et al. Appl. Environ. Microbiol. 64:2497-2502, 1998). An antibodyor fragment thereof could also be prepared using in silico design(Knappik et al., J. Mol. Biol. 296: 57-86, 2000, which is incorporatedherein by reference). In addition or instead of an antibody, the assaymay employ another type of recognition element, such as a receptor orligand binding molecule. Such a recognition element may be a syntheticelement like an artificial antibody or other mimetic. U.S. Pat. Nos.6,255,461; 5,804,563; 6,797,522; 6,670,427; and 5,831,012; and U.S.Patent Application 20040018508; and Ye and Haupt, Anal Bioanal Chem.378: 1887-1897, 2004; Peppas and Huang, Pharm Res. 19: 578-587 2002,provide examples of such synthetic elements and are incorporated hereinby reference. In some instances, antibodies, recognition elements, orsynthetic molecules that recognize a cognate may be available from acommercial source, e.g., Affibody® affinity ligands (Abcam, Inc.Cambridge, Mass. 02139-1517; U.S. Pat. No. 5,831,012, incorporated herein by reference).

Polypeptide Ligands

In some instances, tubular nanostructures may be modified with one ormore ligands that are cellular receptors that recognize and/or bind tobacteria. For example, CD14, which is normally associated withmonocyte/macrophages is known to bind lipopolysaccharide associated withgram negative bacteria as well as lipoteichoic acid associated with thegram positive bacteria Bacillus subtilis (see, e.g., Fan, et al. (1999)Infect. Immun. 67: 2964-2968). Other examples of cellular receptorsinclude, but are not limited to, adenylate cyclase (Bordatellapertussis), Gal alpha 1-4Gal-containing isoreceptors (E. coli),glycoconjugate receptors (enteric bacteria), Lewis(b) blood groupantigen receptor (Heliobacter pylori), CR3 receptor, protein kinasereceptor, galactose N-acetylgalactosamine-inhibitable lectin receptor,and chemokine receptor (Legionella), annexin I (Leishmania mexicana),ActA protein (Listeria monocytogenes), meningococcal virulenceassociated Opa receptors (Meningococcus), alpha5beta3 integrin(Mycobacterium avium-M), heparin sulphate proteoglycan receptor, CD66receptor, integrin receptor, membrane cofactor protein, CD46, GM1, GM2,GM3, and CD3 (Neisseria gonorrhoeae), KDEL receptor (Pseudomonas),epidermal growth factor receptor (Samonella typhiurium), alpha5beta1integrin (Shigella), and nonglycosylated J774 receptor (Streptococci)(see, e.g., U.S. Patent Application 2004/0033584 A1). In some instancesthe pathogen specific receptor/ligand may be bound to the surface of themodified red blood cell through an antibody linkage (see, e.g., U.S.Patent Application 2006/0018912 A1, each incorporated herein byreference).

In some instances, tubular nanostructures may be modified with one ormore ligands that are peptide hormones which interact with specificcognates, for example, cell surface receptors on target cells. Examplesof peptide hormones that may be used to modify tubular nanostructuresinclude, but are not limited to, neuropeptides, for example,enkephalins, neuropeptide Y, somatostatin, corticotropin-releasinghormone, gonadotropin-releasing hormone, adrenocorticotropic hormone,melanocyte-stimulating hormones, bradykinins, tachykinins,cholecystokinin, vasoactive intestinal peptide (VIP), substance P,neurotensin, vasopressin, and calcitonin; cytokines, for example,interleukins (e.g., IL-1 through IL-35), erythropoietin, thrombopoietin,interferon (IFN), granulocyte monocyte colony-stimulating factor(GM-CSF), tumor necrosis factor (TNF), and others; chemokines, e.g.,RANTES, TARC, MIP-1, MCP, and others; growth factors, for example,platelet derived growth factor (PDGF), transforming growth factor beta(TGFβ), nerve growth factor (NGF), epidermal growth factor (EGF),insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF);other peptide hormones, for example, atrial natriuretic factor, insulin,glucagon, angiotensin, prolactin, oxyocin, and others. In one aspect,Mattson, et al., describe functionalizing carbon nanotubes with nervegrowth factor (see U.S. Pat. No. 6,670,179, which is incorporated hereinby reference). Similarly, Liu, et al., describe functionalizing carbonnanotubes with cyclic arginine-glycine-aspartic acid (RGD) peptide, thelatter of which is a ligand for integrin alpha_(v)-beta₃ receptorsup-regulated in a wide range of solid tumors (Liu, et. al., ACS Nano1:50-56, 2007, which is incorporated herein by reference).Alternatively, novel peptides that bind selective target, for example,tumor cells may be generated using phage display methodologies (see,e.g., Spear, et al., Cancer Gene Ther. 8:506-511, 2001, which isincorporated herein by reference).

Small Chemical Compound Ligands

In some aspects, the tubular nanostructure may be configured to includeone or more small chemical compound ligands. As such, a tubularnanostructure may be modified with a small chemical compound ligand thatinteracts with a cognate on a target cell, such as a receptor. Examplesof small chemical compound ligands include, but are not limited to,acetylcholine, adenosine triphosphate (ATP), adenosine, androgens,dopamine, endocannabinoids, epinephrine, folic acid, gamma-aminobutyricacid (GABA), glucocorticoids, glutamate, histamine, leukotrienes,mineralocorticoids, norepinephrine, prostaglandins, serotonin,thromoxanes, or vitamins. For example, the modification of carbonnanotubes with folic acid provides the modified nanotubes which can bindto folate receptors overexpressed on some tumor cells (see Kam et al.,Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005, which is incorporatedherein by reference).

Aptamer Ligands

In some instances, tubular nanostructures may be modified with one ormore ligands that are aptamers. Aptamers are artificial oligonucleotides(DNA or RNA) that can bind to a wide variety of entities (e.g., metalions, small organic molecules, proteins, and cells) with highselectivity, specificity, and affinity. Aptamers may be isolated from alarge library of 10¹⁴ to 10¹⁵ random oligonucleotide sequences using aniterative in vitro selection procedure often termed “systematicevolution of ligands by exponential enrichment” (SELEX; see, e.g., Cao,et al., Current Proteomics 2:31-40, 2005; Proske, et al., Appl.Microbiol. Biotechnol. 69:367-374, 2005, which are incorporated hereinby reference). For example, an RNA aptamer may be generated againstleukemia cells using a cell based SELEX method (see, e.g., Shangguan, etal., Proc. Natl. Acad. Sci. USA 103:11838-11843, 2006, which isincorporated herein by reference). Similarly, an aptamer that recognizesbacteria may be generated using the SELEX method against whole bacteria(see, e.g., Chen, et al., Biochem. Biophys. Res. Commun. 357:743-748,2007, which is incorporated herein by reference).

Lectin Ligands

In some embodiments, tubular nanostructures may be modified with one ormore ligands that are lectins. The term “lectin” was originally used todefine agglutinins which could discriminate among types of red bloodcells and cause agglutination. Currently, the term “lectin” is used moregenerally and includes sugar-binding proteins from many sourcesregardless of their ability to agglutinate cells. Lectins have beenfound in plants, viruses, microorganisms and animals. Because of thespecificity that each lectin has toward a particular carbohydratestructure, even oligosaccharides with identical sugar compositions canbe distinguished or separated. Some lectins will bind only to structureswith mannose or glucose residues, while others may recognize onlygalactose residues. Some lectins require that the particular sugar is ina terminal non-reducing position in the oligosaccharide, while otherscan bind to sugars within the oligosaccharide chain. Some lectins do notdiscriminate between a and b anomers, while others require not only thecorrect anomeric structure but a specific sequence of sugars forbinding. Examples of lectins include, but are not limited to, algallectins, e.g., b-prism lectin; animal lectins, e.g., tachylectin-2,C-type lectins, C-type lectin-like proteins, calnexin-calreticulin,capsid protein, chitin-binding protein, ficolins, fucolectin, H-typelectins, I-type lectins, sialoadhesin, siglec-5, siglec-7, micronemalprotein, P-type lectins, pentrxin, b-trefoil, galectins, congerins,selenocosmia huwena lectin-I, Hcgp-39, Ym1; bacterial lectins, e.g.,Pseudomonas PA-IL, Burkholderia lectins, chromobacterium CV-IIL,Pseudomonas PA IIL, Ralsonia RS-ILL, ADP-ribosylating toxin, Ralstonialectin, Clostridium hemagglutinin, botulinum toxin, tetanus toxin,cyanobacterial lectins, FimH, GafD, PapG, Staphylococcal enterotoxin B,toxin SSL11, toxin SSL5; fungal and yeast lectins, e.g., Aleuriaaurantia lectin, integrin-like lectin, Agaricus lectin, Sclerotiumlectin, Xerocomus lectin, Laetiporus lectin, Marasmius oreadesagglutinin, agrocybe galectin, coprinus galectin-2, Ig-like lectins,L-type lectins; plant lectins, e.g., alpha-D-mannose-specific plantlectins, amaranthus antimicrobial peptide, hevein, pokeweed lectin,Urtica dioica UD, wheat germ agglutinins (WGA-1, WGA-2, WGA-3),artocarpin, artocarpus hirsute AHL, banana lectin, Calsepa, heltuba,jacalin, Maclura pomifera MPA, MornigaM, Parkia lectins, abrin-a, abrusagglutinin, amaranthin, castor bean ricin B, ebulin, mistletoe lectin,TKL-1, cyanovirin-N homolog, and various legume lectins; and virallectins, e.g., capsid protein, coat protein, fiber knob, hemagglutinin,and tailspike protein (see, e.g., E. Bettler, R. Loris, A. Imberty“3D-Lectin database: A web site for images and structural information onlectins” 3rd Electronic Glycoscience Conference, The internet and WorldWide Web, 6-17 Oct. 1997.

Pore-Forming Ligands

In some aspects, tubular nanostructures may be modified with one or moreligands that are pore-forming toxins. Examples of pore-forming toxinsinclude, but are not limited to, beta-pore-forming toxins, e.g.,hemolysin, Panton-Valentine leukocidin S, aerolysin, Clostridialepsilon-toxin; binary toxins, e.g., anthrax, C. perfringens lota toxin,C. difficile cytolethal toxins; cholesterol-dependent cytolysins;pneumolysin; small pore-forming toxins; and gramicidin A

In some aspects, tubular nanostructures may be modified with one or moreligands that are pore-forming antimicrobial peptides. Antimicrobialpeptides represent an abundant and diverse group of molecules that arenaturally produced by many tissues and cell types in a variety ofinvertebrate, plant and animal species. The amino acid composition,amphipathicity, cationic charge and size of antimicrobial peptides allowthem to attach to and insert into microbial membrane bilayers to formpores leading to cellular disruption and death. More than 800 differentantimicrobial peptides have been identified or predicted from nucleicacid sequences, a subset of which have are available in a publicdatabase (see, e.g., Wang & Wang Nucleic Acids Res. 32:D590-D592, 2004),which is incorporated herein by reference). More specific examples ofantimicrobial peptides include, but are not limited to, anionicpeptides, e.g., maximin H5 from amphibians, small anionic peptides richin glutamic and aspartic acids from sheep, cattle and humans, anddermcidin from humans; linear cationic alpha-helical peptides, e.g.,cecropins (A), andropin, moricin, ceratotoxin, and melittin frominsects, cecropin P1 from Ascaris nematodes, magainin (2), dermaseptin,bombinin, brevinin-1, esculentins and buforin II from amphibians,pleurocidin from skin mucous secretions of the winter flounder,seminalplasmin, BMAP, SMAP (SMAP29, ovispirin), PMAP from cattle, sheepand pigs, CAP18 from rabbits and LL37 from humans; cationic peptidesenriched for specific amino acids, e.g., praline-containing peptidesincluding abaecin from honeybees, praline- and arginine-containingpeptides including apidaecins from honeybees, drosocin from Drosophila,pyrrhocoricin from European sap-sucking bug, bactenicins from cattle(Bac7), sheep and goats and PR-39 from pigs, praline- andphenylalanine-containing peptides including prophenin from pigs,glycine-containing peptides including hymenoptaecin from honeybees,glycine- and praline-contining peptides including coleoptericin andholotricin from beetles, tryptophan-containing peptides includingindolicidin from cattle, and small histidine-rich salivary polypeptides,including histatins from humans and higher primates; anionic andcationic peptides that contain cysteine and from disulfide bonds, e.g.,peptides with one disulphide bond including brevinins, peptides with twodisulfide bonds including alpha-defensins from humans (HNP-1, HNP-2,cryptidins), rabbits (NP-1) and rats, beta-defensins from humans (HBD1,DEFB118), cattle, mice, rats, pigs, goats and poultry, and rhesustheta-defensin (RTD-1) from rhesus monkey, insect defensins (defensinA); and anionic and cationic peptide fragments of larger proteins, e.g.,lactoferricin from lactoferrin, casocidin 1 from human casein, andantimicrobial domains from bovine alpha-lactalbumin, human hemoglobin,lysozyme, and ovalbumin (see, e.g., Brogden, Nat. Rev. Microbiol.3:238-250, 2005, which is incorporated herein by reference).

Ligands as Therapeutic Agents

In some instances, the tubular nanostructure as described herein may beconfigured to include one or more ligands that is a therapeutic agent.As such, the one or more therapeutic agent may contribute to disruptionand/or death of the targeted cell in addition to the disruptivepore-forming capability of the tubular nanostructure. Examples oftherapeutic agents that might be incorporated into the tubularnanostructure to aide in disrupting and/or killing cancer cells ormicrobes include anti-cancer therapeutic agents and/or antimicrobialtherapeutic agents. Antimicrobial therapeutic agents may include, butare not limited to, antibacterial, antifungal and antiparasital agents.

Anti-Cancer Therapeutic Agents

In one aspect, the therapeutic agent is an anti-cancer drug. Theanti-cancer drug may be selected from a variety of known small chemicalcompound pharmaceuticals. Alternatively, the chemotherapy agent mayinclude, but is not limited to, an inactivating peptide nuclei acid(PNA), an RNA or DNA oligonucleotide aptamer, short double-stranded RNA(e.g., interfering RNA, microRNA), a peptide, or a protein. Examples ofchemotherapy agents include, but are not limited to, antimetabolitessuch as capecitabine, cladribine, cytarabine, fludarabine,5-fluorouracil, gemcitabine, 6-mercaptopurine, methotrexate, pemetrexed,and 6-thioguanine; antitumor antibiotics such as bleomycin,epipodophyllotoxins such as etoposide and teniposide; taxanes such asdocetaxel and paclitaxel; vinca alkaloids such as vinblasine,vinfristine, and vinorelbine; alkylating agents such as busulfan,carmustine, cyclophosphamide, dacarbazine, ifosfamide, lomustine,mechlorethamine, melphalan, temozolomide, and thiotepa; anthracyclinessuch as daunorubucin, doxorubicin, epirubicin, idarubicin, andmitoxantrope; antitumor antibiotics such as dactinomycin and mitomycin;camptothecins such as irinotecan and topotecan; and platinum analogssuch as carboplatin, cisplatin, and oxaliplatin; hormonally activeagents such as flutamide, bicalutamide, nilutamide, tamoxifen, megestrolacetate, hydrocortisone, prednisone, goserelin acetate, leuprolide,aminoglutethimide, anastrozole, exemestane, and letrozole; andmiscellaneous drugs used for cancer chemotherapy such as arsenictrioxide, erlotinib, gefitinib, imatinib, bortezomib, hydroxyurea,mitoxantrone, retinoic acid derivatives, estramustine, leucovorin andthe photosensitizer Photofrin.

The anti-cancer drug may be a biological agent, e.g., a peptide, aprotein, an enzyme, a receptor and/or an antibody. Examples ofbiological agents currently used to treat cancer include, but are notlimited to, cytokines such as interferon-α, interferon-γ, andinterleukin-2, an enzyme such as asparaginase, and monoclonal antibodiessuch as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, andtrastuzumab.

Novel biological agents for the treatment of cancer may be generated byscreening a peptide phage library, for example, in proliferation assaysagainst cancerous cells, e.g., cultured transformed cells lines and/oragainst primary tumors from patients with various cancers (see, e.g.,Spear, et al. Cancer Gene Therapy 8:506-511, 2001; Krag, et al. CancerRes. 66:7724-7733, 2006, which are incorporated herein by reference).

Antimicrobial Therapeutic Agents

In another aspect, the therapeutic agent is an antibacterial drug.Examples of antibacterial drugs include, but are not limited to,beta-lactam compounds such as penicillin, methicillin, nafcillin,oxacillin, cloxacillin, dicloxacilin, ampicillin, ticarcillin,amoxicillin, carbenicillin, and piperacillin; cephalosporins andcephamycins such as cefadroxil, cefazolin, cephalexin, cephalothin,cephapirin, cephradine, cefaclor, cefamandole, cefonicid, cefuroxime,cefprozil, loracarbef, ceforanide, cefoxitin, cefmetazole, cefotetan,cefoperazone, cefotaxime, ceftazidine, ceftizoxine, ceftriaxone,cefixime, cefpodoxime, proxetil, cefdinir, cefditoren, pivoxil,ceftibuten, moxalactam, and cefepime; other beta-lactam drugs such asaztreonam, clavulanic acid, sulbactam, tazobactam, ertapenem, imipenem,and meropenem; other cell wall membrane active agents such asvancomycin, teicoplanin, daptomycin, fosfomycin, bacitracin, andcycloserine; tetracyclines such as tetracycline, chlortetracycline,oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline,and tigecycline; macrolides such as erythromycin, clarithromycin,azithromycin, and telithromycin; aminoglycosides such as streptomycin,neomycin, kanamycin, amikacin, gentamicin, tobramycin, sisomicin, andnetilmicin; sulfonamides such as sulfacytine, sulfisoxazole,silfamethizole, sulfadiazine, sulfamethoxazole, sulfapyridine, andsulfadoxine; fluoroquinolones such as ciprofloxacin, gatifloxacin,gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, andofloxacin; antimycobacteria drugs such as isoniazid, rifampin,rifabutin, rifapentine, pyrazinamide, ethambutol, ethionamide,capreomycin, clofazimine, and dapsone; and miscellaneous antimicrobialssuch as colistimethate sodium, methenamine hippurate, methenaminemandelate, metronidazole, mupirocin, nitrofurantoin, polymyxin B,clindamycin, choramphenicol, quinupristin-dalfopristin, linezolid,spectrinomycin, trimethoprim, pyrimethamine, andtrimethoprim-sulfamethoxazole.

In another aspect, the therapeutic agent is an antifungal agent.Examples of antifungal agents include, but are not limited to,anidulafungin, amphotericin B, butaconazole, butenafine, caspofungin,clotrimazole, econazole, fluconazole, flucytosine griseofulvin,itraconazole, ketoconazole, miconazole, micafungin, naftifine,natamycin, nystatin, oxiconazole, sulconazole, terbinafine, terconazole,tioconazole, tolnaftate, and/or voriconazole.

In another aspect, the therapeutic agent is an anti-parasite agent.Examples of anti-parasite agents include, but are not limited to,antimalaria drugs such as chloroquine, amodiaquine, quinine, quinidine,mefloquine, primaquine, sulfadoxine-pyrimethamine, atovaquone-proguanil,chlorproguanil-dapsone, proguanil, doxycycline, halofantrine,lumefantrine, and artemisinins; treatments for amebiasis such asmetronidazole, iodoquinol, paromomycin, diloxanide furoate, pentamidine,sodium stibogluconate, emetine, and dehydroemetine; and otheranti-parasite agents such as pentamidine, nitazoxanide, suramin,melarsoprol, eflornithine, nifurtimox, clindamycin, albendazole, andtinidazole.

In some instances, the antimicrobial agent may be an antimicrobialpeptide. A number of naturally occurring antimicrobial peptides havebeen described herein and amino acid sequence information for a subsetof these may be found as part of a public database (see, e.g., Wang &Wang Nucleic Acids Res. 32:D590-D592, 2004), which is incorporatedherein by reference). Alternatively, a phage library of random peptidesmay be used to screen for peptides with antimicrobial properties againstlive bacteria, fungi and/or parasites. The DNA sequence corresponding toan antimicrobial peptide may be generated ex vivo using standardrecombinant DNA and protein purification techniques and subsequentlyattached to tubular nanostructures using the methods described herein.

Markers on Tubular Nanostructures

In some instances, the tubular nanostructure as described herein may beconfigured to include one or more marker. The one or more marker mayinclude, e.g., a fluorescent marker, a radioactive marker, a quantumdot, a contrast agent for magnetic resonance imaging (MM) marker, orcombinations thereof. One or more markers may be used to facilitateimaging of the tubular nanostructure in association with target cells ororganelles.

Fluorescent Markers

In one aspect, the tubular nanostructure may include one or more markerscapable of fluorescence in response to appropriate wavelengths ofelectromagnetic energy. The one or more fluorescent marker associatedwith the tubular nanostructure may include one or more of thefluorescent compounds currently approved by the United States Food andDrug Administration (FDA) for use in human mammals including, but notlimited to, fluorescein (FITC), indocyanine green, and rhodamine B.FITC, for example, may be readily added to a carbon nanotubefunctionalized with PL-PEG-NH₂ as described in Kam, et al., Proc. Natl.Acad. Sci. USA 102:11600-11605, 2005, which is incorporated herein byreference. Alternatively, the one or more fluorescent marker associatedwith the tubular nanostrucure may include one or more of a number ofother fluorescent compounds including, but not limited to, cyanine dyessuch as Cy5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, N.J.,USA) and/or a variety of Alexa Fluor dyes including Alexa Fluor 633,Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680,Alexa Fluor 700 and Alexa Fluor 750 (Molecular Probes-Invitrogen,Carlsbad, Calif., USA; see, e.g., U.S. Pat. App. No. 2005/0171434 A1).Additional fluorophores include IRD41 and IRD700 (LI-COR, Lincoln,Nebr., USA), NIR-1 and 1C5-OSu (Dejindo, Kumamotot, Japan), LaJolla Blue(Diatron, Miami, Fla., USA), FAR-Blue, FAR-Green One, and FAR-Green Two(Innosense, Giacosa, Italy), ADS 790-NS and ADS 821-NS (American DyeSource, Montreal, Calif.) and VivoTag 680 (VT680; VisEn Medical, Woburn,Mass., USA). Many of these fluorophores are available from commercialsources either attached to primary or secondary antibodies or asamine-reactive succinimidyl or monosuccinimidyl esters, for example,ready for conjugation to appropriately functionalized tubularnanostructures using the methods described herein. Alternatively, thefluorophore may be added to a small single-stranded DNA and thefluorophore/DNA conjugate attached to the tubular nanostructure vianon-covalent interaction between the DNA and nanotube (see, e.g., Kam,et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005, which isincorporated herein by reference).

In one aspect, the tubular nanostructure may include one or more markersthat are quantum dots (Q-dots). Q-dots are nanocrystal semiconductorswith unique optical properties, fluorescing at various excitationwavelengths depending upon composition and size. A variety of Q-dots areavailable from a number of commercial sources and may be added totubular nanostructures through, e.g., amines, carboxyl groups, biotin,streptavidin, secondary antibodies, and phopholipid-PEG (from, e.g.,Evident Technologies, Troy, N.Y.; Invitrogen, Carlsbad, Calif.). Forexample, Chen et al., describe adding Q-dots conjugated to streptavidinto nanotubes modified with biotin through pyrene bound to the nanotubeside-wall via π-π stacking (see Chen et al., Proc. Natl. Acad. Sci. USA104:8218-8222, 2007, which is incorporated herein by reference.Similarly, Didenko and Baskin describe using an enzymatic process withhorseradish peroxidase to add streptavidin conjugated Q-dots tonanotubes (BioTechniques 40:295-302, 2006, which is incorporated hereinby reference).

In a further embodiment, the tubular nanostructures themselves may beinherently fluorescent at specific wavelengths of electromagneticenergy. For example, single-walled carbon nanotubes have been shown toexhibit photoluminescence in the near infrared when excited by a diodelaser at 785 nm (see, e.g., Welsher, et al., Nano Lett 8: 586-590, 2008,which is incorporated herein by reference).

Fluorescence associated with tubular nanostructures may be monitoredusing invasive and non-invasive methods. Invasive methods areexemplified by insertion of an endoscope or a catheter containingoptical fibers for fluorescence excitation and measurement into bodycavities or vessels (see, e.g., U.S. Pat. Nos. 7,341,557; 6,389,307,which are incorporated herein by reference). Non-invasive methods areexemplified by fluorescence mediated molecular tomography. For example,non-invasive monitoring of near infrared (NIR) fluorescence may beperformed using fluorescence mediated molecular tomography as describedin U.S. Pat. No. 6,615,063, which is incorporated herein by reference.Additional information regarding NIR imaging in human subjects isdescribed in Frangioni Curr. Op. Chem. Biol. 7:626-634, 2003, which isincorporated herein by reference. In some instances, a wireless systemmay be used in which light sources such as light emitting diodes (LEDs)of appropriate wavelength as well as detectors such as charge-coupleddevices (CCDs) are housed along with a power supply and a wirelesscommunication circuit to create a device that may be placed on the skinof a subject to monitor NIR signal as described by Muehlemann, et al.,Optics Express, 16:10323, 2008, which is incorporated herein byreference.

Radioactive Markers

In another embodiment, the tubular nanostructure may include one or moremarkers that are radioactive. Tubular nanostructures modified with oneor more radioisotopes may be monitored using a gamma camera, positronemission tomography (PET), other gamma ray probe. Examples ofradioactive molecular that might be used for this purpose include, butare not limited to, carbon-11, nitrogen-13, oxygen-15, fluorine-18,rubidium-82, yttrium-86, technetium-99, iodine-123, indium-111,thallium-201. For example, indium-111 may be added to carbon nanotubesusing bifunctional metal chelating agents such as2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA-NCS) or diethylentriaminepentaacetic (DTPA) (see, e.g.,McDevitt, et al., J. Nucl. Med. 48:1180-1189, 2007; Singh, et al., Proc.Natl. Acad. Sci. USA 103:3357-3362, 2006, which are incorporated hereinby reference). Similar methods are described for adding yttrium-86 tocarbon nanotubes (McDevitt et al., PLoS ONE 2:e907, 2007, which isincorporated herein by reference).

Contrast Agent Markers

In another aspect, the tubular nanostructures may include one or moremarkers that are contrast agents used in magnetic resonance imaging(MRI). For example, tubular nanostructures, e.g., carbon nanotubes maybe combined with the high-spin paramagnetic gadolinium (Gd⁺) metal ionsto form an effective contrast agent for MRI (see, e.g., Sitharaman &Wilson Int. J. Nanomed. 1:291-295, 2006, which is incorporated herein byreference). Alternatively, tubular nanostructures may be functionalizedwith a combination of iron and cobalt salts to form MRI and nearinfrared imaging agents (see, e.g., Seo, et al., Nat. Mater. 5:971-976,2006, which is incorporated herein by reference). Other divalent metalions that might be included in tubular nanostructures for MRI detectioninclude, but are not limited to, cobalt, nickel, zinc, magnesium, andmanganese (see, e.g., U.S. Patent Application 2008/0124281, which isincorporated herein by reference). Alternatively, bacterial derivedmagnetic nanocrystals may be absorbed onto the tubular nanostructure asdescribed in U.S. Patent Application 2007/0200085, which is incorporatedherein by reference.

Activated Markers on Tubular Nanostructures

The tubular nanostructure as described herein may include one or moremarkers that may be activated. One or more markers associated with thetubular nanostructure may be activated by a ligand reaction, anchoringin the membrane and interaction with a hydrophobic medium, and/or changein the cellular environment (e.g., changes in pH). One or more markerassociated with the tubular nanostructure may be activated upon reachingthe intended target. Alternatively, one or more marker associated withthe tubular nanostructure may be activated upon disruption and/or deathof the target cell. Alternatively, one or more marker associated withthe tubular nanostructure may be activated upon passage of the tubularnanostructure from one cellular compartment to another.

Ligand Reaction Activated Markers

The one or more activatable marker associated with the tubularnanostructure may be activated by a ligand reaction. The marker may beactivated when the marker or a component associated with the markerbinds to, comes in close contact with, or otherwise interacts with aligand associated with the target cell or organelle. The marker mayinclude a donor and an acceptor molecule that undergo fluorescenceresonance energy transfer (FRET) in response to interaction of themarker with the ligand. FRET is a distance-dependent interaction betweenthe electronic excited states of two dye molecules in which excitationis transferred from a donor molecule to an acceptor molecule withoutemission of a photon. In some instances, interaction of a donor moleculewith an acceptor molecule may lead to a shift in the emission wavelengthassociated with excitation of the acceptor molecule. In other instances,interaction of a donor molecule with an acceptor molecule may lead toquenching of the donor emission.

The donor and acceptor molecules of the marker may be conjugated to thesame biomolecule such that changes in the conformation of thebiomolecule in response to ligand interaction move the donor andacceptor molecules relative to one another. Examples of biomoleculesthat might be used in this manner include, but are not limited to,polynucleotides, e.g., aptamers or polypeptides, e.g., antibodies. Inthis instance, the aptamers or antibodies associated with the marker maybe the same aptamer or antibody used to bind the tubular nanostructureto cognates on a target cell or organelle. Alternatively, the aptamersor antibodies associated with the marker may be distinct, interactingwith different components on the target cell or organelle. Otherbiomolecules that change conformation in response to binding a ligandmay be used for this purpose.

Alternatively, the donor and acceptor molecules may be conjugated toseparate biomolecules such that changes in proximity of the separatebiomolecules moves the donor and acceptor molecules relative to oneanother. In this instance, the target cell or organelle may be modifiedwith either a donor or acceptor molecule while the tubular nanostructuremay be modified with the corresponding donor or acceptor molecule. Ineither instance, the interaction of the tubular nanostructure with thetarget cell or organelle triggers a measurable response.

Tubular nanostructures may be modified with one or more activatablemarker, for example, an aptamer-based molecular beacon. Molecularbeacons are dual labeled aptamer probes with a donor fluorophore at oneend and an acceptor fluorophore or quencher at the other end. Uponbinding of a specific target, the aptamer undergoes a conformationalshift such that the distance between the donor fluorophore and theacceptor fluorophore or quencher is altered, leading to a change inmeasurable fluorescence through the phenomenon of FRET, as discussedabove (see, e.g. Cao, et al., Current Proteomics, 2:31-40, 2005, whichis incorporated herein by reference). In some instances, thefluorescence associated with aptamer may be quenched until the tubularnanostructure reaches its intended target. Alternatively, thefluorescence associated with the aptamer may be shifted in wavelengthwhen the tubular nanostructure reaches its intended target.

Tubular nanostructures may be modified with one or more activatablemarker that is an antibody-based molecular beacon. In this instance, theantibody may be labeled with a donor or acceptor molecule and asecondary protein associated with the antibody such as Protein A,Protein G, or a F_(ab) fragment, for example, may be labeled with thecorresponding donor or acceptor molecule (see, e.g., Lichlyter, et al.,Biosens. Bioelectron. 19:219-226, 2003, which is incorporated herein byreference). Interaction of the labeled antibody/secondary proteincomplex with the appropriate ligand shifts the antibody and thesecondary protein relative to one another and induces a FRET signal.Alternatively, the one or more marker may be an antibody labeled nearthe antigen-binding site with a donor or acceptor molecule and aflexible arm attached to an analog of the antigen recognized by theantibody which itself includes the corresponding donor or acceptormolecule (see, e.g. U.S. Patent Application 2006/0172318 A1).Competition for the antigen-binding site by the analog and the actualligand on the target cell or organelle results in measurable changes inthe spatial relationship between the donor and acceptor molecules. Insome instances, the tubular nanostructures may be modified with a markerthat is an antibody that is labeled with a solvent sensitivefluorophore, e.g., dansyl chloride(5-dimethylaminonaphthalene-1-sulfonyl chloride), and exhibits a shiftin fluorescent signal in response to interaction with a ligandassociated with the target cell or organelle antigen (see, e.g., BrennanJ. Fluor. 9:295-312, 1999, which is incorporated herein by reference).An antibody of this type may be labeled such that interaction of theligand with the antibody shields the solvent sensitive fluorescent inthe active binding site from the solvent water, in a measurable changefluorescence intensity (see, e.g., Bright, et al. Anal. Chem.62:1065-1069, 1990, which is incorporated herein by reference).

The donor and acceptor fluorophore pairs associated with the marker mayinclude, but are not limited to, fluorescein and tetramethylrhodamine;IAEDANS and fluorescein; fluorescein and fluorescein; and BODIPY FL andBODIPY FL. Alternatively, the marker may include any of a number ofAlexa Fluor (AF) fluorophores (from, e.g., Invitrogen, Carlsbad, Calif.)paired with other AF fluorophores for use in FRET. Some examples includeAF 350 with AF 488; AF 488 with AF 546, AF 555, AF 568, or AF 647; AF546 with AF 568, AF 594, or AF 647; AF 555 with AF594 or AF647; AF 568with AF6456; and AF594 with AF 647.

Alternatively, the donor and acceptor fluorophore pairs associated withthe marker may include cyanine dyes. The cyanine dyes Cy3, Cy5, Cy5.5and Cy7, which emit in the red and far red wavelength range (>550 nm),offer a number of advantages for FRET-based detection systems. Theiremission range is such that background fluorescence is often reduced andrelatively large distances (>100 Å) can be measured as a result of thehigh extinction coefficients and good quantum yields. For example, Cy3,which emits maximally at 570 nm and Cy5, which emits at 670 nm, may beused as a donor-acceptor pair. When the Cy3 and Cy5 are not proximal toone another, excitation at 540 nm results only in the emission of lightby Cy3 at 590 nm. In contrast, when Cy3 and Cy5 are brought intoproximity by a conformation change in an aptamer, for example,excitation at 540 nm results in an emission at 680 nm.

Alternatively, the donor or acceptor molecular of the marker may includeone or more semiconductor quantum dots (Q-dots) paired with anappropriate organic dye donor or acceptor molecule as described byBawendi, et al., in U.S. Pat. No. 6,306,610, which is incorporatedherein by reference.

In some instances, the donor molecule of the marker may be a quenchingdye that quenches the fluorescence of visible light-excited fluorophoreswhen in close proximity. Examples include DABCYL, the non-fluorescingdiarylrhodamine derivative dyes QSY 7, QSY 9 and QSY 21 (from, e.g.,Invitrogen, Carlsbad, Calif.), the non-fluorescing Black Hole QuenchersBHQ0, BHQ1, BHQ2, and BHQ3 (from, e.g., Biosearch Technologies, Inc.,Novato, Calif., USA) and Eclipse (from, e.g., Applera Corp., Norwalk,Conn., USA). A variety of donor fluorophore and quencher pairs may beconsidered for FRET including but not limited to fluorescein withDABCYL; EDANS with DABCYL; or fluorescein with QSY 7 and QSY 9. Ingeneral, QSY 7 and QSY 9 dyes efficiently quench the fluorescenceemission of donor dyes including blue-fluorescent coumarins, green- ororange-fluorescent dyes, and conjugates of the Texas Red and Alexa Fluor594 dyes. QSY 21 dye efficiently quenches all red-fluorescent dyes. Anumber of the Alexa Fluor (AF) fluorophores (from, e.g., Invitrogen,Carlsbad, Calif.) may be paired with quenching molecules as follows: AF350 with QSY 35 or DABCYL; AF 488 with QSY 35, DABCYL, QSY7 or QSY9; AF546 with QSY 35, DABCYL, QSY7 or QSY9; AF 555 with QSY7 or QSY9; AF 568with QSY7, QSY9 or QSY21; AF 594 with QSY21; and AF 647 with QSY 21.

In some instances, the tubular nanostructure itself may act as aquencher. Carbon nanotubes, for example, can act collectively asquenchers of covalently tethered and/or π stacked pyrenes andchromophores. This phenomenon is attributed to electron transfer orenergy transfer from the photoactive compound to the carbon nanotubes ifsufficiently close in proximity. As such, fluorescence emitted bychromophores bound to carbon nanotubes may be quenched by theassociation. For example, lysophospholipid1,2-dipalmitoyl-sn-glycero-3-lysophosphoethanolamine-N-(Liss-aminerhodamine B sulfonyl), or Rd-LPE may be added to carbon nanotubes asdescribed by Lin et al. (Appl. Phys. Lett. 89:143118, 2006, which isincorporated herein by reference). In this instance, Rd-LPE solubilizescarbon nanotubes in aqueous solution via pure hydrophobic interactionsand these self-assembled supramolecular complexes, once excited, readilyundergo fluorescence energy transfer from the Rd-LPE to the carbonnanotubes, quenching the rhodamine associated fluorescence. This energytransfer may be used to detect membrane translocation of modified carbonnanotubes and dissociation of Rd-LPE in cells, for example. Duringtranslocation through the plasma membrane, the lipid-rhodamine moietymay be transferred off the carbon nanotubes and as such the quenching isremoved and the rhodamine associated fluorescence is detected.Alternatively, the lipid rhodamine moiety is stripped from the carbonnanotube during entry into the cell, quenching is removed and rhodamineassociated fluorescence is detected.

Lipid translocation in combination with carbon nanotubes crossing themembrane is accompanied by lipid flip or lipid flip-flop within thelipid bilayer membrane.

Lipid Membrane Reactive Markers

The one or more activatable marker associated with the tubularnanostructure may be activated by a lipid versus aqueous environment. Assuch, incorporation of the tubular nanostructure modified with anactivatable marker that is lipid sensitive into the lipid bilayer of atarget tissue or organelle may result in a measurable response. Forexample, the marker may be a fluorescent dye such as one of severalaminonaphthlethenyl-pyridinium (ANEP) dyes which are essentiallynon-fluorescent in an aqueous environment but fluoresce within a lipidenvironment. Examples of lipid sensitive fluorescent ANEP dyes include,but are not limited to, di-4ANEPPS and di-8-ANEPPS. When bound tophospholipid vesicles, di-8-ANEPPS has excitation/emission maxima of˜467/631 nm. The fluorescence excitation/emission maxima of di-4-ANEPPSbound to neuronal membranes, for example, are ˜475/617 nm.

Alternatively, the marker may be a derivative of nitrobenzoxadiazole(NBD) which is almost non-fluorescent in aqueous solvents. The NBDfluorophore is moderately polar and both its homologous 6-carbon and12-carbon fatty acid analogs and the phospholipids derived from theseprobes may be used to sense the lipid-water interface region ofmembranes.

The marker may be fluorescent phospholipid analog β-DPH HPC whichcomprises diphenylhexatriene propionic acid coupled toglycerophosphocholine at the sn-2 position. DPH and its derivativesexhibit strong fluorescence enhancement when incorporated intomembranes, as well as sensitive fluorescence polarization (anisotropy)responses to lipid ordering. β-DPH HPC may be used to specifically labelone leaflet of a lipid bilayer, thus facilitating analysis of membraneasymmetry.

A number of phospholipid analogs with pyrene-labeled sn-2 acyl chains,e.g.,4-hydroxy-N,N,N-trimethyl-10-oxo-7-((1-oxo-10-(1-pyrenyl)decyl)oxy)-hydroxideare also non-fluorescent in aqueous solution but become fluorescent in alipid environment. Various pyrenedecanoyl-labeled glycerophospholipidsmay be used for this purpose including but not limited to those withphosphocholine, phosphoglycerol, and phosphomethanol head groups.

Alternatively, the marker may be a derivative of the polyunsaturatedfatty acid cis-parinaric acid which offers several experimentallyadvantageous optical properties, including a very large fluorescenceStokes shift (˜100 nm) and an almost complete lack of fluorescence inwater.

Cell Environment Reactive Markers

The one or more activatable marker associated with the tubularnanostructure may be activated in response to the cellular environment.For example, the marker may be activated by changes pH and/or byenzymatic reactions associated with lipid bilayer and/or components ofthe cytoplasm.

The tubular nanostructures may include a marker that is sensitive to pHchanges in the cellular environment. For example, the marker may be a pHsensitive fluorescent dye such as LysoSensor Yellow/Blue DND-160(Invitrogen, Carlsbad, Calif.) which undergoes a pH dependent emissionand excitation shift to longer wavelengths in acidic environments.Examples of pH sensitive dyes include, but are not limited to, otherLysoSensor probes, e.g., LysoSensor Blue DND-167 and LysoSensor GreenDND-189 which are almost nonfluorescent except when inside acidiccompartments; and fluorescein containing dyes such asdichlorofluorescein, carboxydichlorofluorescein,carboxydifluorofluorescein, and BCECF; and Oregon Green 514 carboxylicacid, Oregon Green 488 carboxylic acid, 5-(and 6-)carboxy-2′,7′-,9-amino-6-chloro-2-methoxyacridine (ACMA) (e.g., from Invitrogen,Carlsbad, Calif.).

The tubular nanostructures may include a marker that is activated by achemical process. For example, the marker may be a bis-BODIPY FL C₁₁-PCwhich has BODIPY FL dye-labeled sn-1 and sn-2 acyl groups, resulting inpartially quenched fluorescence that increases when one of the acylgroups is hydrolyzed by phospholipase A₁ or A₂. The phospholipase may beassociated with either the membrane or the cytoplasm. The hydrolysisproducts are BODIPY FL undecanoic acid and BODIPY FL dye-labeledlysophosphatidylcholine. Other examples include markers that are linkedto the tubular nanostructures through a cleavable disulfide bond, esterlinkage, or ortho carboxy phenol derived acetal linkage (see, e.g., U.S.Pat. Nos. 7,087,770 and 7,348,453, which are incorporated herein byreference). For example, Q-dots linked to carbon nanotubes by disulfidebond may be cleaved from the nanotubes upon entry into the cell (see,e.g., Chen, et al., Proc. Natl. Acad. Sci. USA 104:8218-8222, 2007,which is incorporated herein by reference). As such, donor and acceptormolecules associated with the marker may be separated from one anotherby breaking a cleavable bond, resulting in a measurable signal.

Assemblies of Tubular Nanostructures

The one or more tubular nanostructures as described herein may beindividual, discrete nanotubes. Alternatively, tubular nanostructuresmay form higher order assemblies or compositive tubular nanostructures.A composite tubular nanostructure may comprise two or more tubularnanostructures each including a hydrophobic surface region, eachhydrophobic region flanked by two hydrophilic surface regions configuredto form a pore in a lipid bilayer membrane. Composite tubularnanostructures may be used to create multiple pores at one or more sitesin the targeted lipid bilayer.

In general, carbon nanotubes, for example, have a tendency to formlarge, insoluble aggregates due to substantial van der Waalsinteractions. As such, solubilization techniques may be used to break upthese aggregates into smaller bundles and/or individual nanotubes. Thenanotubes may be solubilized by acid oxidation, by surfactants, bypolymer wrapping and/or by chemical functionalization, for example.Solubilization in acid or surfactant or other solubilizing agent such aspolyoxometalates, for example, may be carried out in the presence ofsonication and may be monitored using scanning and/or transmissionelectron microscopy (see, e.g., Fei, et al., Nanotechnol. 17:1589-1593,2006, which is incorporated herein by reference). Alternatively, Ramanspectroscopy may be used to monitor disaggregation of carbon nanotubes.For example, Raman signals at 266 cm⁻¹ correspond to aggregated nanotubebundles whereas a broad photoluminescence peak observed at approximately3,200 cm⁻¹ (1,050 nm) corresponds to individual tubes (see, e.g., Kam,et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005, which isincorporated herein by reference). There is evidence to suggest thatelectron and ion irradiation of nanotubes give rise to covalent bondsbetween tubes in bundles (see, e.g., Sammalkorpi, et al., Nucl. Instr.Methods Phys. Res. B 228:142-145, 2005; Szabados, et al., Phys. Rev.73:195404, 2006, which are incorporated herein by reference).

In a further aspect, bundles of two or more tubular nanostructures maybe formed by modification of the nanotube sidewall that confersattraction between individual nanotubes. For example, bundles of two ormore tubular nanostructures may be formed by combining an appropriateratio of nanotubes modified with biotin and nanotubes modified withstreptavidin. Other biomolecule binding interactions that might be usedto construct composite tubular nanostructures include, but are notlimited to, protein-protein interactions, antibody-antigen interactions,sense-antisense DNA or RNA interactions, aptamer-target interaction,peptide-nucleic acid (PNA)-DNA or RNA interactions. Biomolecules for usein forming higher ordered bundles of tubular nanostructures may be addedto the nanotubes using one or more of the methods described herein.Optionally, asymmetric sidewall functionalization in which one surfaceor portion of a surface is masked during the functionalization processmay be used to selectively place biomolecules on the surface of tubularnanostructures as described herein. As such, the compatible surfaces areexpected to come together to form composite tubular nanostructures.

Two or more tubular nanostructures may be bundled together through theinteraction of biomolecules associated with the nanotubes that normallyoligomerize into higher order complexes. Tubular nanostructures may bemodified with a protein or proteins that naturally form a triplex, forexample, and as such would bring together three associated nanotubes. Anexample is the ATP responsive, cation-selective ion channels P2X1, P2X2,and P2X3 which have been shown by various means including atomic forcemicroscopy to form trimeric structures (see, e.g., Barrera, et al., J.Biol. Chem. 280:10759-10765, 2005, which is incorporated herein byreference). Alternatively, tubular nanostructures may be modified with aprotein or proteins that naturally form a heptamer and as such wouldbring together seven associated nanotubes. An example is thepore-forming toxin hemolysin which forms a heptameric beta-barrelstructure in biological membranes.

Assembly of Tubular Nanostructures Enabling Active Transport,Facilitated Transport, or Passive Transport

Tubular nanostructures as described herein may be further modified tocontrol flow of biomolecules through the pores formed by the nanotubesin the lipid bilayer. For example, tubular nanostructures may bemodified with one or more proteins or peptides that facilitate activeand or passive transport across the pore. Active transport requires anexternal energy source, e.g., the hydrolysis of ATP to transportbiomolecules such as ions against a concentration gradient, thebiomolecules moving, for example, from low to high concentration. Incontrast, passive transport is driven by the concentration gradient ofthe biomolecule across an open pore, the biomolecules moving from highto low concentration to establish equilibrium. Facilitated transport isa form of passive transport in which materials are moved across theplasma membrane by a transport protein down their concentrationgradient; hence, it does not require energy. Biomolecules that areinvolved in active active transport, facilitated transport, or passivetransport of molecules across the lipid bilayer may be incorporated intothe tubular nanostructures.

Tubular nanostructures may be include one or more components of anATP-binding cassette transporters (ABC transporters). ABC transportersare composed of transmembrane domains connected to one or more ligandbinding domains on either the intracellular or extracellular side of thelipid bilayer and one or more ATP binding domains on the intracellularsurface. ATP transporters may be classified as half or fulltransporters. Full transporters may contain two transmembrane domainsand two ATP binding domains and are fully functional. Half transporterscontain one transdomain and one ATP binding domain and must combine withanother half transporter to be fully functional. As such, a tubularnanostructure may include all or part of a full transporter sufficientto confer functionality. Alternatively, a tubular nanostructure mayinclude half of a full transporter or all of part of a half transporterwhich upon interacting with one or more similarly modified tubularnanostructure generates a functional ABC transporter.

One or more tubular nanostructures may include all or part of an ABCtransporter, for example, the cystic fibrosis transmembrane conductanceregulator (CFTR), the transporter associated with antigen processing(TAP), or the multidrug resistance efflux pump (MDR). There are sevendistinct gene families of ABC transporters found in humans including,but not limited to, ABCA, ABCB, ABCD, ABCE, ABCF, and ABCG, with eachfamily consisting of 1 to 12 members. Examples of ABC transporter genesfound in prokaryotes include, but are not limited to, transporters suchas Carbohydrate Uptake Transporter-1 (CUT1), Carbohydrate UptakeTransporter-2 (CUT2), Polar Amino Acid Uptake Transporter (PAAT),Peptide/Opine/Nickel Uptake Transporter (PepT), Hydrophobic Amino AcidUptake Transporter (HAAT), Sulfate/Tungstate Uptake Transporter (SulT),Phosphate Uptake Transporter (PhoT), Molybdate Uptake Transporter(MolT), Phosphonate Uptake Transporter (PhnT), Ferric Iron UptakeTransporter (FeT), Polyamine/Opine/Phosphonate Uptake Transporter(POPT), Quaternary Amine Uptake Transporter (QAT), Vitamin B12 UptakeTransporter (B12T), Iron Chelate Uptake Transporter (FeCT),Manganese/Zinc/Iron Chelate Uptake Transporter (MZT),Nitrate/Nitrite/Cyanate Uptake Transporter (NitT), Taurine UptakeTransporter (TauT), Cobalt Uptake Transporter (CoT), Thiamin UptakeTransporter (ThiT). Brachyspira Iron Transporter (BIT), Siderophore-Fe3+Uptake Transporter (SIUT), Nickel Uptake Transporter (NiT),Nickel/Cobalt Uptake Transporter (NiCoT), and Methionine UptakeTransporter (MUT); and exporters such as Lipid Exporter (LipidE),Capsular Polysaccharide Exporter (CPSE), Lipooligosaccharide Exporter(LOSE), Lipopolysaccharide Exporter (LPSE), Teichoic Acid Exporter(TAE), Drug Exporter-1 (DrugE1), Lipid Exporter (LipidE), Putative HemeExporter (HemeE), β-Glucan Exporter (GlucanE), Protein-1 Exporter(Prot1E), Protein-2 Exporter (Prot2E), Peptide-1 Exporter (Pep1E),Peptide-2 Exporter (Pep2E), Peptide-3 Exporter (Pep3E), ProbableGlycolipid Exporter (DevE), Na+ Exporter (NatE), Microcin B17 Exporter(McbE), Drug Exporter-2 (DrugE2), Microcin J25 Exporter (McjD),Drug/Siderophore Exporter-3 (DrugE3), (Putative) Drug ResistanceATPase-1 (Drug RA1), (Putative) Drug Resistance ATPase-2 (Drug RA2),Macrolide Exporter (MacB), Peptide-4 Exporter (Pep4E), 3-componentPeptide-5 Exporter (Pep5E), Lipoprotein Translocase (LPT), β-Exotoxin IExporter ((βETE), AmfS Peptide Exporter (AmfS-E), SkfA Peptide Exporter(SkfA-E), and CydDC Cysteine Exporter (CydDC-E).

Alternatively, the tubular nanostructures may include one or morecomponents of an ion channel. Ion channels are integral membraneproteins that regulate the flow of ions across the cell membrane andoften include a circular arrangement of identical or homologous proteinsclosely packed around a water-filled pore through the plane of the lipidbilayer. The pore-forming subunit(s) are called the α subunit, while theauxiliary subunits are denoted β, γ, and so on. In some ion channels,passage through the pore is governed by a “gate,” which may be opened orclosed by chemical or electrical signals, temperature, or mechanicalforce, depending on the variety of channel. Examples of ion channelsthat might be incorporated into one or more tubular nanostructuresinclude, but are not limited to, voltage-gated sodium, calcium andpotassium channels, voltage gated proton channels, transient receptorpotential channels (TRP), cyclic nucleotide-gated channels, light gatedchannels, inward-rectifier potassium channels, calcium-activatedpotassium channels, and ligand gated channels, e.g., ionotropicglutamate-gated receptors, ATP-gated P2X receptors, and anion-permeablegamma-aminobutyric acid-gated GABA receptors.

In some instances, the tubular nanostructures may include one or morecomponents that alone or in combination form a synthetic ion channel.Compounds that might be used to form synthetic ion channels include, butare not limited to, crown ethers, octiphenyl derivatives, octa- anddecapeptides, and bolaamphiphiles (two-headed amphiphiles; see, e.g.,Fyles Chem. Soc. Rev. 36: 335-347, 2007, which is incorporated herein byreference).

In some instances, opening or closing of the pore associated with thetubular nanostructure may be controlled by a component of the tubularnanostructure that reversibly covers and or uncovers the one or morepore openings. For example, the tubular nanostructures may include oneor more components at one or both pore openings that change inconformation in response stimuli such as, for example, pH, temperature,electric field, light, and or ligand binding. Conformational changes inproteins, for example, in response to stimuli may modulate activity ofthe protein and or play a role in signal transduction. An example is theglutamate receptor family of glutamate binding proteins in which theglutamate binding domain is in a clam-shell like hinge region whichopens in the absence of glutamate and closes in the presense ofglutamate (see, e.g., Dinglehine, et al., Pharmacol. Rev. 51:7-62, 1999,which is incorporated herein by reference). Similarly, DNA and RNAbiomolecules such as aptamers, for example, may be designed to change inconformation in response to ligand binding (see, e.g., Ha, et al., PNAS96:9077-9082, 1999, which is incorporated herein by reference). As such,the tubular nanostructure may be modified with a biomolecule such as aprotein or an aptamer at one or both pore openings that is able to openand close in response to ligand binding and as such can control the flowof other biomolecules through the pore.

Alternatively, the tubular nanostructures may include one or morecomponents at one or both pore openings that is responsive to light orelectromagnetic energy. Electromagnetic energy may include gamma rays,x-rays, ultraviolet, visible, infrared, microwave and or radio waves. Inthis instance, the one or more component may contain one or morecleavage sites, for example, that are activated by electromagneticenergy and results in removal of portion of the component that may becovering the pore opening. For example, Rock, et al., describe a numberof dithiane adduct derivatives that may be used with proteins asphotolabile linkers (U.S. Pat. No. 5,767,288, which is incorporatedherein by reference). Alternatively, the energy activated component maychange conformation in response to electromagnetic energy and as suchcover or uncover the pore opening.

In a further aspect, the tubular nanotubes may include components thatare magnetic and allow binding of one or both ends of the pore to amagnet bead that physically blocks the pore opening. For example, one orboth ends of the tubular nanostructure may be modified with moleculeshaving magnetic properties. Examples of molecules having magneticproperties include but are not limited to the common magnetic metalsiron, nickel, and cobalt and their alloys as well as the rare earthmetals and alloys or combinations thereof such as for examplegadolinium, samarium, and europium. Tubular nanostructures such ascarbon nanotubes, for example, may be functionalized with iron and orgadolinium, for example, using methods described in Seo, et al., (Nat.Mater. 5:971-976, 2006) and Sitharaman & Wilson (Int. J. Nanomed.1:291-295, 2006), respectively, which are incorporated herein byreference. The magnetized tubular nanotubes may be administered to asubject to form pores in targeted lipid membranes, and magnetic beadsadministered at a subsequent time point to block the pore opening.Alternatively, the magnetized tubular structures may be combined withmagnetic beads prior to administration, and an external magnetic source,for example, may be used to separate the beads from the nanotubes.

In some instances, the pore associated with the tubular nanostructuremay be covered by a nanoparticle such as for example a bead which hasbeen modified with an aptamer or antibody, for example, that binds to acorresponding ligand at one or both ends of the tubular nanostructure.Alternatively, the nanoparticle may include streptavidin or biotin whichbinds to biotin or streptavidin, respectively, at the end of the tubularnanostructure.

The tubular nanostructures may be further modified to allow forcontrolled release of an agent such as, for example, a therapeutic agentand or toxin in proximity to the pore opening. For example, the tubularnanotubes may include a binding moiety such as an aptamer or antibodysituated at one or both ends of the tubular nanostructure to which isreversibly bound an agent. The affinity of the antibody for the agent issuch that the agent dissociates from the antibody and because of itsproximity to the pore, has a higher probability of passing through thepore. Alternatively, the tubular nanostructure may include a ligand thatis recognized by a bifunctional binding moiety such as, for example, abifunctional antibody. In this instance, the bifunctional antibody has acomponent that binds to a ligand on the tubular nanostructure as well asa component that reversibly binds to an agent such as, for example, atherapeutic agent and or toxin. In this instance, the bifunctionalantibody carrying an agent may be administered to the subject at a pointin time following administration of the tubular nanostructures. As such,the tubular nanostructure embedded into the lipid bilayer, binds thebifunctional antibody, and over time, the agent is released from thebifunctional antibody and passes through the lipid bilayer by way of theproximal tubular nanostructure pore.

Tubular Nanostructure Directed to Specific Organelles

In some instances, the tubular nanostructures as described herein may bemodified in such a manner as to allow transit of the nanotubes throughthe plasma membrane with subsequent targeting and insertion into thelipid bilayer of one or more internal organelles. Once targeted to thelipid bilayer of the organelle membrane, the tubular nanostructure mayform pores that enable active transport, facilitated transport, orpassive transport of contents into or out of the organelle. In certainorganelles, disruption of the lipid bilayer may lead to cell death. Inone example, the membrane target is the outer membrane of mitochondria.In general, mitochondrial outer membrane permeabilization is consideredthe “point of no return” during apoptosis of cells as it results in thediffusion to the cytosol of numerous proteins that normally reside inthe space between the outer and inner mitochondrial membranes andinitiates a cascade of events leading to cell death (see, e.g., Chipuk,et al., Cell Death Differ. 13:1396-1402, 2006, which is incorporatedherein by reference). As such, tubular nanostructures may be selectivelydirected to the outer membrane of mitochondria in target cells wherethey insert into and disrupt the outer mitochondrial membrane leading totarget cell death.

The tubular nanostructures with hydrophobic surface region flanked bytwo hydrophilic surface regions for insertion and retention in a lipidbilayer may be modified in such a manner as to mask the hydrophilic endsand allow transit through the plasma membrane. In one embodiment, thehydrophilic ends of the tubular nanostructure are modified with ahydrophobic moiety through a chemical bond that may be cleaved once thenanotube has passed into the cell. Examples of biologically cleavablebonds include, but are not limited to, disulfide bonds, diols, diazobonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enolesters, enamines and imines (see, e.g., U.S. Pat. Nos. 7,087,770,7,098,030 and 7,348,453, which are incorporated herein by reference).

Alternatively, the cleavable bond may be a photolabile bond. Examples ofhydrophobic moieties that might be added to the ends of the tubularnanostructure include, but are not limited to, non-polar hydrocarbonchains of various lengths. In one aspect, the hydrophobic moiety is anester that can be cleaved by an intracellular esterase to form ahydrophilic acid moiety and alcohol moiety. For example, hydrophilicmoieties may be masked by acetoxymethyl esters of phosphates, sulfates,or other compounds having alcohol moieties or acid moieties which willenhance permeability of the tubular nanostructure across the lipidbilayer membrane. Because acetoxymethyl esters are rapidly cleavedintracellularly, they facilitate the delivery of tubular nanostructuresinto the cytoplasm of the cell without puncturing or disruption of thecell plasma membrane (see, e.g., Schultz et al., J. Biol. Chem. 268:6316-6322, 1993, which are incorporated herein by reference). Oncewithin the cytoplasm, the tubular nanostructures having a hydrophobicsurface region flanked by two hydrophilic surface regions is configuredto form a pore in the lipid bilayer membrane of the cellular organelle.

Alternatively, the tubular nanostructure may be tethered to a proteintransduction domain (PTD) such as human immunodeficiency virus type 1(HIV-1) transactivator of transcription (Tat), Drosophila Antennapedia(Antp), or herpes simplex virus VP22 that masks the hydrophilic ends andfacilitates entry of the nanotubes into the cell. In one aspect, all orpart of the 86 amino acid long Tat protein may be added to tubularnanostructures through primary amines associated with the peptide and/orthe functionalized nanotubes using the methods described herein (alsosee, e.g., Santra, et al., Chem. Commun. 24:2810-2811, 2004, which isincorporated herein by reference). The Tat protein or other proteintransduction domain may be linked to the tubular nanostructure to thehydrophilic regions on either end of the nanotube through a cleavablebond such as those described herein and as such removed from the tubularnanostructure once the latter has entered the cell, unmasking thehydrophilic regions.

Under certain conditions, the masked tubular nanostructures may beactively taken up by the cell through the process of endocytosis (see,e.g., Kam, et al., Angew. Chem. Int. Ed. 44:1-6, 2005, which isincorporated herein by reference). Endocytosis is the process wherebycells absorb extracellular material by engulfing the material with theircell membrane. The engulfed material is contained in small vesicles thatpinch off from the plasma membrane, enter the cytoplasm and fuse withother intracellular vesicles, e.g., endosomes or lysosomes.

Material such as tubular nanostructures may be released from endosomesby a number of mechanisms. In one aspect, artificial acceleration ofendosomal release may be achieved by photo-excitation of fluorescentprobes associated with the engulfed material (see, e.g., Matsushita, etal., FEBS Lett. 572:221-226, 2004, which is incorporated herein byreference). Alternatively, the tubular nanostructure may include a pHsensitive element that is activated in the low pH environment of theendosome. In a further aspect, all or part of the influenza virushemagglutinin-2 subunit (HA-2), a pH-dependent fusogenic peptide thatinduces lysis of membranes at low pH, may be used to induce efficientrelease of encapsulated material from cellular endosomes (see, e.g.,Yoshikawa, et al., J. Mol. Biol. 380:777-782, 2008, which isincorporated herein by reference).

Alternatively, the masked tubular nanostructures may enter the cell bypassing directly through the cell membrane and into the cytoplasm. Inthis instance, the tubular nanostructure may include moieties on thesurface of the nanotubes that confers direct passage through the lipidbilayer, e.g., an amphiphilic striated surface on the nanotube. Thedeposition of a hydrophilic-hydrophobic striated pattern of molecules,e.g., the anionic ligand 11-mercapto-1-undecanesulphonate (MUS) and thehydrophobic ligand 1-octanethiol (OT) on the surface of nanotubes mayfacilitate direct passage of the tubular nanostructures into thecytoplasm (see, e.g., Verma, et al., Nature Materials_7: 588-95, 2008,which is incorporated herein by reference). Once the masked tubularnanostructures has entered the cytoplasm, it can be modified to revealtubular nanostructures with hydrophobic surface region flanked by twohydrophilic surface regions and at least one ligand bound to thenanostructure and configured to bind one or more cognates on anorganellar membrane, e.g., a mitochondrial membrane.

The one or more tubular nanostructures may include one or more ligandsthat binds to one or more cognate on a cellular organelle, e.g.,mitochondria, as well as one or more ligand that binds to one or morecognates on the cell surface membrane of the target cell. The one ormore ligands may be an antibody, antibody-coated liposome,polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein,carbohydrate, lipid, toxin, lectin, or any combination thereof asdescribed herein. Cognates associated with a mitochondrial membrane mayinclude at least one of a protein, a carbohydrate, a glycoprotein, aglycolipid, a sphingolipid, a glycerolipid, or metabolites thereof.Examples of cognates associated with the mitochondrial outer membrane,for example, include, but are not limited to, carnitine palmitoyltransferase 2, translocase of outer membrane (TOM70), sorting/assemblymachinery, ANT, voltage dependent anion channel (VDAC/Porin), andmonoamine oxidase. In some instances, one or more tubular nanostructuresmay include one or more ligands that bind to one or more cognates on theinner mitochondrial membrane. A cognate of the inner mitochondrialmembrane may be a membrane associated receptor or protein, e.g., one ormore proteins associated with the carnitine acyltransferase IItransporter, NADH dehydrogenase complex (Complex I), succinatedehydrogenase (Complex II), cytochrome bc1 complex (Complex III),cytochrome c oxidase complex (Complex IV), ATP synthase, or uncouplingprotein (UCP).

Pharmaceutical Formulation of a Tubular Nanostructure and Administrationto a Subject

The compositions and methods described herein for inserting a tubularnanostructure into a lipid bilayer membrane are useful for treatment ofa disease or condition, e.g., cancer or infectious disease, in amammalian subject in need thereof. A pharmaceutical formulationincluding the tubular nanostructures or the composite tubularnanostructures described herein may be formulated neat or may becombined with one or more acceptable carriers, diluents, excipients,and/or vehicles such as, for example, buffers, surfactants,preservatives, solubilizing agents, isotonicity agents, and stablilizingagents as appropriate. A “pharmaceutically acceptable” carrier, forexample, may be approved by a regulatory agency of the state and/orFederal government such as, for example, the United States Food and DrugAdministration (US FDA) or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans. Conventional formulation techniques generallyknown to practitioners are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000), which is incorporated herein by reference.

Acceptable pharmaceutical carriers include, but are not limited to, thefollowing: sugars, such as lactose, glucose and sucrose; starches, suchas corn starch and potato starch; cellulose, and its derivatives, suchas sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate,and hydroxymethylcellulose; polyvinylpyrrolidone; cyclodextrin andamylose; powdered tragacanth; malt; gelatin, agar and pectin; talc;oils, such as mineral oil, polyhydroxyethoxylated castor oil, peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; polysaccharides, such as alginic acid and acacia; fattyacids and fatty acid derivatives, such as stearic acid, magnesium andsodium stearate, fatty acid amines, pentaerythritol fatty acid esters;and fatty acid monoglycerides and diglycerides; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;buffering agents, such as magnesium hydroxide, aluminum hydroxide andsodium benzoate/benzoic acid; water; isotonic saline; Ringer's solution;ethyl alcohol; phosphate buffer solutions; other non-toxic compatiblesubstances employed in pharmaceutical compositions.

A pharmaceutical formulation including the tubular nanostructures or thecomposite tubular nanostructures described herein may be formulated in apharmaceutically acceptable liquid carrier. The liquid carrier orvehicle may be a solvent or liquid dispersion medium comprising, forexample, water, saline solution, ethanol, a polyol, vegetable oils,nontoxic glyceryl esters, and suitable mixtures thereof. The solubilityof a chemical blocking agent may be enhanced using solubility enhancerssuch as, for example, water; diols, such as propylene glycol andglycerol; mono-alcohols, such as ethanol, propanol, and higher alcohols;DMSO (dimethylsulfoxide); dimethylformamide, N,N-dimethylacetamide;2-pyrrolidone, N-(2-hydroxyethyl) pyrrolidone, N-methylpyrrolidone,1-dodecylazacycloheptan-2-one and othern-substituted-alkyl-azacycloalkyl-2-ones and othern-substituted-alkyl-azacycloalkyl-2-ones (azones). The proper fluiditymay be maintained, for example, by the formation of liposomes, by themaintenance of the necessary particle size in the case of dispersions orby the use of surfactants. One or more antimicrobial agent may beincluded in the formulation such as, for example, parabens,chlorobutanol, phenol, sorbic acid, and/or thimerosal to preventmicrobial contamination. In some instances, it may be preferable toinclude isotonic agents such as, for example, sugars, buffers, sodiumchloride or combinations thereof.

A pharmaceutical formulation including the tubular nanostructures or thecomposite tubular nanostructures described herein may be formulated fortransdermal delivery. For example, water-insoluble, stratumcorneum-lipid modifiers such as for example 1,3-dioxanes, 1,3-dioxolanesand derivatives thereof, 5-, 6-, 7-, or 8-numbered lactams (e.g.,butyrolactam, caprolactam), morpholine, cycloalkylene carbonate havebeen described for use in transdermal iontophoresis (see, e.g., U.S.Pat. No. 5,527,797, which is incorporated herein by reference). Othersuitable penetration-enhancing agents include but are not limited toethanol, hexanol, cyclohexanol, polyethylene glycol monolaurate,azacycloalkan-2-ones, linoleic acid, capric acid, lauric acid,neodecanoic acid hexane, cyclohexane, isopropylbenzene; aldehydes andketones such as cyclohexanone, acetamide; N,N-di(lower alkyl)acetamidessuch as N,N-diethylacetamide, N,N-dimethyl acetamide;N-(2-hydroxyethyl)acetamide; esters such as N,N-di-lower alkylsulfoxides; essential oils such as propylene glycol, glycerine,isopropyl myristate, and ethyl oleate; salicylates; and mixtures of anyof the above (see, e.g., U.S. Patent Publication 2008/0119449).

In some instances, the pharmaceutical formulation including the tubularnanostructures or the composite tubular nanostructures described hereinmay be formulated in a dispersed or dissolved form in a hydrogel orpolymer associated with, for example, implantable or a transdermaldelivery method. Examples of hydrogels and/or polymers include but arenot limited to gelled and/or cross-linked water swellable polyolefins,polycarbonates, polyesters, polyamides, polyethers, polyepoxides andpolyurethanes such as, for example, poly(acrylamide),poly(2-hydroxyethyl acrylate), poly(2-hydroxypropyl acrylate),poly(N-vinyl-2-pyrrolidone), poly(n-methylol acrylamide), poly(diacetoneacrylamide), poly(2-hydroxylethyl methacrylate), poly(allyl alcohol).Other suitable polymers include but are not limited to cellulose ethers,methyl cellulose ethers, cellulose and hydroxylated cellulose, methylcellulose and hydroxylated methyl cellulose, gums such as guar, locust,karaya, xanthan gelatin, and derivatives thereof. For iontophoresis, forexample, the polymer or polymers may include an ionizable group such as,for example, (alkyl, aryl or aralkyl) carboxylic, phosphoric, glycolicor sulfonic acids, (alkyl, aryl or aralkyl) quaternary ammonium saltsand protonated amines and/or other positively charged species asdescribed in U.S. Pat. No. 5,558,633, which is incorporated herein byreference in its entirety.

Information regarding formulation of FDA approved tubular nanostructuresor the composite tubular nanostructures may be found in the packageinsert and labeling documentation associated with each approved agent. Acompendium of package inserts and FDA approved labeling may be found inthe Physician's Desk Reference. For those tubular nanostructures orcomposite tubular nanostructures described herein which do not currentlyhave a formulation appropriate for use in any of the delivery methodsdescribed above, an appropriate formulation may be determinedempirically and/or experimentally using standard practices. Thepharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Pharmaceutical compositions including the tubular nanostructures or thecomposite tubular nanostructures described herein can be administered toan individual by any of a number of routes including, but not limitedto, oral, nasal, pulmonary, rectal, transdermal, vaginal, ortransmucosal routes as well as the parenteral routes. Suitableparenteral delivery routes for the pharmaceutical compositions include,but are not limited to, intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections.Examples of microbead and nanoparticle approaches and materials thatwould be appropriate for the delivery of pharmaceutical compositionsincluding the tubular nanostructures or the composite tubularnanostructures are described in Nanomaterials for Medical Diagnosis andTherapy, 1^(st) edition, edited by Challa Kumar (Nanotechnologies forthe Life Sciences Vol. 10, 2007, WILEY-VCH Verlag GmbH & Co. KGaA,Wienham; Nanomaterials for Cancer Therapy, edited by Challa Kumar(Nanotechnologies for the Life Sciences, Vol. 6, 2006, WILEY-VCH VerlagGmbH & Co. KGaA, Wienham, which are incorporated herein by reference).

The methods and compositions are further described with reference to thefollowing examples; however, it is to be understood that the methods andcompositions are not limited to such examples.

Exemplary Aspects

Example 1

Preparation of Tubular Nanostructures for Targeting Cancer Cells

One or more tubular nanostructures may be used to selectively target andkill tumor cells in a subject with cancer. The one or more tubularnanostructures may be selectively directed to the tumor cells through aligand associated with the tubular nanostructures that recognizes acorresponding cognate on the membrane of the tumor cells. The one ormore ligands may be at least a portion of an antibody, antibody-coatedliposome, polynucleotide, polypeptide, receptor, viral plasmid, polymer,protein, carbohydrate, lipid, toxins, pore-forming toxins, or lectin.The one or more cognates on a membrane of a tumor cell may be a leastone of a protein, a carbohydrate, a glycoprotein, a glycolipid, asphingolipid, a glycerolipid, or a metabolite thereof. For example, oneor more carbon nanotubes may be modified with a ligand that is anantibody or fragment thereof that specifically binds a cognate that is acell surface receptor on a tumor cell. A tumor cell may be a breastcancer cell. An example of a cell surface receptor on a breast cancercell may be the HER2/erb/neu receptor. An antibody to HER2 may beattached to tubular nanostructures and used to direct interaction of thecarbon nanotube to the breast cancer cells. Once at the targeted tumorcell, the one or more tubular nanostructures may form pores in theplasma membrane through which intracellular and extracellular componentsmay flow. Disruption of the highly controlled barrier function of theplasma membrane ultimately results in death of the targeted tumor cell.

One or more tubular nanostructures for selective targeting of tumorcells may be derived from one or more carbon nanotubes. Carbon nanotubesmay be generated using one of several methods including, but not limitedto, arc-discharge, laser ablation, chemical vapor deposition (CVD), orthe gas-phase catalytic process (HiPCO). For example, carbon nanotubesmay be generated using an appropriate carbon source as described hereinin the presence of one or more Group VI and/or Group VIII transitionmetals, e.g., chromium, iron, cobalt, ruthenium, nickel and platinumusing laser vaporization with dual pulsed lasers as described in U.S.Pat. No. 7,008,604, which is incorporated herein by reference.Alternatively, carbon nanotubes may be purchased from a commercialsource (from, e.g., Unidym, Menlo Park, Calif.; Sigma-Aldrich, St.Louis, Mo.; Carbolex, Inc., Lexington, Ky.)

The carbon nanotubes may be used directly for functionalization.Alternatively, the carbon nanotubes may be cut to generate more uniform,open-ended nanotubes. Disrupting the closed ends of the carbon nanotubewill also facilitate functionalization of the ends. Carbon nanotubes maybe cut by any of a number of different methods as described herein. Forexample, carbon nanotubes may be cut using an ultra microtome (Wang etal., Nanotechnology 18: 055301, 2007, which is incorporated herein byreference). In this instance, a magnetic field may be used to align thenanotubes prior to cutting. Pristine nanotubes are dispersed in water,stabilized in surfactant and passed under pressure through a nylonfilter in the bore of a resistive coil magnetic, e.g., with a magnetfield of 17.3 T. The nanotubes aligned on the filter are dried undervacuum. The resulting film of aligned nanotubes is cut and the stripsstacked to form a rigid block of nanotubes. The block of nanotubes maybe cut with a cryo-diamond knife at a temperature of approximately −60°C. using an ultra microtome, e.g., the Leica EM UC6 or EM FC6 microtome(from, e.g., Leica Microsystems, Bannockburn, Ill.).

The carbon nanotubes may be further treated by oxidation to facilitatefunctionalization of the ends and side-walls of the carbon nanotubes. Assuch, carbon nanotubes may be oxidized in the presence of strongoxidizing agents, e.g., nitric acid, KMnO₄/H₂SO₄, O₂, K₂Cr₂O₇/H₂SO₄ orOsO₄, to clean the nanotubes, cut the nanotubes, and/or prepare thenanotubes for functionalization. Oxidation of carbon nanotubes in nitricacid at a temperature of 120° C., for example, may be used to furtherclean the nanotubes by eliminating amorphous carbon and othercontaminants. Oxidation may be also be used to cut the carbon nanotubesinto shorter lengths and to open up the ends of the nanotubes. Inaddition, oxidation creates defects in the carbon nanotube sidewallwhich may be used to add moieties to the otherwise inert sidewall. Assuch, oxidation may be used to prepare the carbon nanotubes forfunctionalization. Following oxidation, the carbon nanotubes may betreated with neutralizing agents and further purified by size usingelectrophoresis, filtration or chromatography.

The carbon nanotubes are inherently hydrophobic. To facilitate improvedinsertion of the tubular nanostructure into the plasma membrane of atumor cell, the carbon nanotubes may be functionalized at either or bothends with hydrophilic moieties. Hydrophilic moieties might include oneor more of amines, amides, charged or polar amino acids, alcohols,carboxylic groups, oxides, ester groups, ether groups, ester-ethergroups, ketones, aldehydes, or derivatives thereof. For example,carboxylic groups may be added to a carbon nanotube by sonicating thecarbon nanotubes in a 3:1 vol/vol solution of concentrated sulfuric acid(98%) and concentrated nitric acid (70%) for 24 hours at 35-40° C., andwashed with water, leaving an open hole in the nanotube andfunctionalizing the open end with one or more carboxyl group (see, e.g.,Li, et al., Proc. Natl. Acad. Sci. USA 103:19658-19663, 2006, which isincorporated herein by reference).

The carbon nanotube may be further modified with a ligand that is anantibody or fragment thereof that specifically binds a cognate that is acell surface receptor on a breast cancer. For example, the carbonnanotube may be modified with an antibody that specifically binds to theHER2/neu receptor on certain breast cancer cells. An example of anantibody that binds HER2/neu receptors on breast cancer cells istrastuzumab (Genentech, South San Francisco, Calif.). An antibody suchas trastuzumab may be added to functionalized carbon nanotubes using oneor more of the methods described herein. Alternatively, any of a numberof commercially available antibodies to the HER2/neu receptor may beused (from, e.g., Novus Biologicals, Littleton, Colo.; AffinityBioReagents, Inc., Golden Colo.; Genway Biotech, Inc., San Diego,Calif.). For example, a thiolated antibody may be conjugated to carbonnanotubes functionalized with primary amines or phospholipid(PL)-PEG-NH₂ (see, e.g., McDevitt, et al., J. Nucl. Med. 48:1180-1189,2007; Welsher, et al., Nano Lett. 8:586-590, 2008, which areincorporated herein by reference). PL-PEG-NH₂ (from, e.g., Avanti PolarLipids, Inc., Alabaster, Ala.) at a concentration of 100-200 μM is mixedwith approximately 0.25 mg/ml carbon nanotubes previously functionalizedwith hydrophilic ends in water and sonicated for 1 hour. The suspensionis centrifuged at 200,000× g for 1 hour and the resulting pelletdiscarded. Excess PL-PEG-NH₂ may be removed by filtration through afilter, e.g., a filter with a 100 kDa molecular weight cut off (from,e.g., Millipore, Billerica, Mass.). The PL-PEG-NH₂ modified carbonnanotubes may be conjugated to thiolated antibody through a sulfo-SMCClinker. Thiolation may be accomplished using 2-iminothiolane.HCl whichreacts with primary amines on the antibody to introduce sulfhydrylgroups. The antibody (10 mg/ml) is mixed with 10-fold molar excess of2-iminothiolane.HCl (e.g., 46 μl of a 14 mM stock solution of2-iminothiolane to each milliliter of antibody solution) in phosphatebuffered saline in the presence of 20 mM EDTA for 2 hours. Unreacted2-iminothiolane may be removed by filtration through a 100 kDa filter.To finish conjugation, the PL-PEG-NH₂ modified carbon nanotubes (400 nM)are treated with 2 mM sulfo-SMCC (Pierce-Thermo Scientific, Rockford,Ill.) for 2 hours in phosphate buffered saline at pH 7.4 and excesssulfo-SMCC removed by filtration as above. The sulfo-SMCC treated carbonnanotubes are mixed with the thiolated antibody at a 1:10 molar ratioand allowed to incubate overnight at 4° C. to generate the carbonnanotube-antibody conjugate.

The tubular nanostructure as described herein is further modified with aligand that is an antibody or fragment thereof, e.g., trastuzumabantibody, that specifically binds a cognate that is a HER2/neu cellsurface receptor on certain breast cancer cells. The tubularnanostructure is targeted to the breast cancer cells, wherein thetubular nanostructure has a hydrophobic surface region flanked by twohydrophilic surface regions and is configured to form a pore in a lipidbilayer membrane of the breast cancer cell, and thus causing cell deathof the breast cancer cell.

Example 2

Tubular Nanostructure with Lectin

One or more tubular nanostructures modified with a lectin may be used toselectively target and kill tumor cells in a subject with cancer. Theone or more tubular nanostructures may be selectively directed to thetumor cells through a ligand, e.g., a lectin, associated with thetubular nanostructures that recognizes a corresponding cognate on themembrane of the tumor cells. In some instances, the binding of thelectin to the cognate on the target tumor cell may contribute todisruption and death of the targeted cell. For example, the lectin maybe one of several galactose-binding plant lectins, e.g., Ricinuscommunis agglutinin I (RCA_(I)) or Bandeirae simplicifolia lectin I,which may bind to abnormally high quantities of galactose moieties foundon the plasma membranes of some tumor cells, such as bladder carcinomacells, and thereby weakening the membrane of the tumor cells andcontributing to cell death (see, e.g., U.S. Pat. No. 4,496,539, which isincorporated herein by reference).

Tubular nanostructures generated using the methods as described hereinmay be modified with a lectin. For example, RCA_(I), which is a 120,000molecular weight protein, may be purchased from commercial sources(e.g., from Sigma-Aldrich, St. Louis, Mo.) and used to functionalizetubular nanostructures. Alternatively, all or part of RCAI may begenerated using standard recombinant molecular biology techniques andcorresponding cDNA sequences reported in GenBank as part of the NationalCenter for Biotechnology Information (NCBI) (see, e.g., Benson, et al.,Nucleic Acids Res. 36:D25-D30, 2008, which is incorporated herein byreference). RCA_(I) may be conjugated to primary amines associated withtubular nanostructures using the methods described herein.

The tubular nanostructures may be further modified with one or moreligand such as an antibody or an aptamer, for example, that directs thenanotubes to the target tissue and enhances target specificity. Forexample, one or more aptamers specific for one or more cognates on atumor cell may be generated using SELEX. In general, a diverse libraryof random DNA oligonucleotide sequences (40 to 55 nucleotides in length)may be amplified using the polymerase chain reaction (PCR) in thepresence of a 5′primer labeled with a fluorescent tag and a 3′ primerlabeled with biotin. After denaturing the DNA under alkaline conditions,the fluorescently labeled sense single strand DNA (ssDNA) can beseparated from the biotinylated anti sense ssDNA using streptavidincoated Sepharose beads. Aptamers to live cells, for example, may beisolated by incubating the fluorescently labeled ssDNA with live cellsand monitoring ssDNA binding by flow cytometry. Those ssDNA sequencesthat bind to the cells may be subjected to another round of PCR in thepresence of labeled primers as described above. This cycle may berepeated several times until aptamers of appropriate binding affinityand selectivity are selected. Once the specific aptamer sequence for atarget has been identified, the oligonucleotide sequence may begenerated using standard procedures.

Example 3

Tubular Nanostructure with Toxin

One or more tubular nanostructures modified with one or more toxins maybe used to selectively disrupt and kill target cells. The one or moretoxins may act as a ligand to direct specific interaction with a cognateon a target cell. Alternatively, the one or more tubular nanostructuresmay be further modified with a ligand that specifically binds to acognate on a target cell and brings the associated one or more toxinsinto proximity with the target cell.

The tubular nanostructures may include one or more toxins thatspecifically target and kill bacteria. For example, the one or moretoxins may be one or more antimicrobial peptides. Antimicrobial peptidesrepresent an abundant and diverse group of molecules that are naturallyproduced by many tissues and cell types in a variety of invertebrate,plant and animal species. The amino acid composition, amphipathicity,cationic charge and size of antimicrobial peptides allow them to attachto and insert into microbial membrane bilayers to form pores leading tocellular disruption and death. Antimicrobial peptides are generated aspart of the host innate immune system and as such are capable ofselectively targeting bacterial cells. For example, magainin 2, anantimicrobial peptide originally isolated from Xenopus laevis, may firstbe attracted to the net negative charges on the surface of bacteriaassociated with anionic phospholipids and the phosphate groups oflipopolysaccharide (LPS) on Gram-negative bacteria and teichoic acids onGram-positive cells. Passing through the outer portions of the bacteria,the magainin 2 reaches the cytoplasmic membrane where it oligomerizeswith other magainin 2 subunits to form a toroidal pore resulting in theimmediate loss of cytoplasmic potassium and cell death (see, e.g.,Brogden Nat. Rev. Microbiol. 3:238-250, 2005, which is incorporatedherein by reference). As such, magainin 2, for example, may be used totarget and contribute to the death of bacteria.

Antimicrobial peptides, e.g., magainin 2 may be added to a tubularnanostructure using the methods described herein. Like manyantimicrobial peptides, magainin 2 is a relatively small peptide withonly 23 amino acids and as such is amenable to direct chemical peptidesynthesis using commercial custom peptide synthesis services (from,e.g., Invitrogen, Carlsbad, Calif.; Sigma-Genosys, The Woodlands, Tex.;Abgent, San Diego, Calif.). Alternatively, magainin 2 or otherantimicrobial peptides may be generated using standard recombinantmolecular biology techniques and DNA sequence information available inGenBank as part of the National Center for Biotechnology Information(NCBI) (Benson, et al., Nucleic Acids Res. 36:D25-D30, 2008, which isincorporated herein by reference). The peptide is preferably synthesizedwith an amino terminal cysteine residue that enables interaction with areactive group associated with the tubular nanostructure such as asuccinimidyl group, for example. Tubular nanostructures such as carbonnanotubes are synthesized as described herein. The nanotubes are furtherfunctionalized with a primary amine group followed by addition ofN-succinimidyl-3-maleimidopropionate (from, e.g., Pierce-ThermoScientific, Rockford, Ill.) in preparation for adding the peptide. Forexample, carbon nanotubes (5-10 mg) are suspended in 2 milliliters ofdimethylformamide (DMF) and mixed with 2 milliliters ofN-succinimidyl-3-maleimidopropionate in DMF. The reaction is stirred for4-8 hours at room temperature and excessN-succinimidyl-3-maleimidopropionate removed by incubation with a resincontaining a primary amine, e.g., PEGA-NH₂ resin (from, e.g.,Sigma-Aldrich, St. Louis, Mo.). The resin is removed by filtration. Thecarbon nanotubes as prepared are added to approximately 4 mg of purifiedpeptides in 1.5 milliliters of an aqueous solution, e.g., water. After4-8 hours, PEGA-NH₂ resin derivatized withN-succinimidyl-3-maleimidopropionate may be used to eliminate excesspeptide and is removed by filtration.

In some instances, the tubular nanostructures may specifically targettumor cells and include one or more toxins. The one or more toxins maybe a pore-forming toxin, e.g., aerolysin. Aerolysin is a bacterial toxinderived from Aeromonas spp that binds toglycosylphosphatidylinositol-anchored proteins (GPI-AP) on mammaliancells and oligomerizes, inserting into the target membranes and formingchannels that cause cell death. Aerolysin may be generated usingstandard recombinant molecular biology techniques and the knownpolynucleotide sequences of aerolysin (see, e.g., Howard, et al., J.Bacteriol. 169:2869-2871, 1987, which is incorporated herein byreference).

The one or more toxin associated with a tubular nanostructure may byitself lack sufficient cell type specificity to selectively target tumorcells, for example. As such, the tubular nanostructures may furtherinclude a ligand that specifically binds a cognate on tumor cells. Forexample, the tubular nanostructures may include the luteinizinghormone-releasing hormone (LHRH) peptide. LHRH binds to LHRH receptorsthat are overexpressed on ovarian tumor cells and to a lesser extent onbreast and prostate tumor cells (see, e.g., Khandare, et al., J.Pharmacol. Exp. Ther. 317:929-937, 2006; Dharap, et al., Proc. Natl.Acad. Sci. USA 102:12962-12967, 2005, which are incorporated herein byreference). LHRH may be generated using standard recombinant molecularbiology techniques and the known polynucleotide sequences of LHRHavailable in GenBank as part of the National Center for BiotechnologyInformation (NCBI) (see, e.g., Benson, et al., Nucleic Acids Res.36:D25-D30, 2008, which is incorporated herein by reference).Alternatively, LHRH may be obtained from commercial sources (from, e.g.,Sigma-Aldrich, St. Louis, Mo.). Alternatively, LHRH may be purified froma nature source. LHRH may be conjugated to tubular nanostructure throughits primary amines using the methods described herein for peptideligands.

Example 4

Tubular Nanostructure with Controlled Flow

One or more tubular nanostructures targeted to a tumor cell may befurther modified to control flow of biomolecules through the poresformed by the nanotubes in the lipid bilayer. For example, the poreassociated with the tubular nanostructure may be closed by physicallyblocking the pore. The pore may be blocked by administering an agent tothe subject that specifically binds at or near the pore opening. Theagent may be a nanoparticle such as, for example a bead. The bead may bemodified with an antibody, for example, that recognizes and binds to aligand associated with one or both ends of the tubular nanostructure.Alternatively, the bead may be modified with a ligand that binds to anantibody associated with one or both ends of the tubular nanostructure.Alternatively, the bead may be modified with either streptavidin orbiotin and as such binds to biotin or streptavidin, respectively,attached to the tubular nanostructure. Other biomolecule bindinginteractions that might be used to bind a bead to a tubularnanostructure include but are not limited to protein-proteininteractions, sense-antisense DNA or RNA interactions, aptamer-targetinteraction, peptide-nucleic acid (PNA)-DNA or RNA interactions. Thebeads may be administered to the subject at some point in time afteradministration of the tubular nanostructures to block further movementof biomolecules through the pore.

Beads may be modified with an antibody, for example, using a number ofmethods. For example, antibodies may be conjugated to beads using amineor carboxyl derivatized beads (from, e.g., Pierce, Rockford, Ill.) usingthe cross linking methods described herein. Alternatively, an antibodymay be conjugated to beads using immunoglobulin binding proteins derivedfrom bacteria such as, for example, Protein A or Protein G. Beadsmodified with Protein A or Protein G are available from commercialsources (e.g., μMACS Protein A or μMACS Protein G MicroBeads, fromMiltenyi Biotec, Auburn, Calif.; Protein A or Protein G sepharose, fromInvitrogen, Carlsbad, Calif.).

Alternatively beads may be labeled with either streptavidin or biotin.Beads labeled with streptavidin are available from commercial sources(from, e.g., Applied Biosystems, Foster City, Calif.; BD Biosciences,San Jose, Calif.; and Invitrogen, Carlsbad, Calif.). Beads labeled withbiotin are also available from commercial sources (from, e.g.,Polysciences, Inc., Warrington, Pa.). Biotin and or streptavidin, forexample, may be added to a tubular nanostructure using the methodsdescribed herein.

In some instances, the interaction between the tubular nanostructure andthe bead may be reversible. For example, the binding affinity of thebead to the tubular nanostructure may be such that over time the twoentities dissociate and the pore is re-opened. Alternatively, the beadmay be dissociated from the tubular nanostructure by competition withfree ligand.

Example 5

Tubular Nanostructure with Controlled Release of an Agent

One or more tubular nanostructures targeted to a tumor cell may befurther modified to allow delivery of an agent proximal to the porethrough the lipid bilayer formed by the nanotube. The agent may be atherapeutic agent and or a toxin that contributes to the death of thetumor cell. The agent may be bound to an antibody or aptamer that isitself bound to the tubular nanostructure. Alternatively, the agent maybe bound to an antibody or aptamer that is administered subsequent toadministration of the tubular nanostructures and binds to the membraneassociated nanotube. In either instance, the agent dissociates from theantibody or aptamer and due to its proximity to the nanotube pore, flowsthrough the pore and through the associated lipid bilayer.

An antibody may be generated against a therapeutic agent using themethods described herein. For example, antibodies to taxols such as thechemotherapy agent paclitaxel, for example, may be generated byattaching the taxol to a carrier protein such as bovine thyroglobin(BTG), immunizing mice, and generating monoclonal antibodies usingstandard hybridoma techniques (see, e.g., U.S. Pat. No. 7,175,993, whichis incorporated herein by reference). Optionally, additional screeningmay be done to access binding affinity for the therapeutic agent toidentify antibodies that have sufficient affinity to bind the agent butare able to dissociate the agent over a given time frame.Antigen/antibody on-off rates may be assessed using a Biacore 3000, forexample (from Biacore, Inc., Piscataway, N.J.). Alternatively anantibody to a therapeutic agent may be available from a commercialsource. For example, antibodies to the chemotherapeutic agentdoxorubicin are commercially available (from, e.g., United StatesBiological, Swampscott, Mass.).

An antibody that recognizes and binds a chemotherapy agent, for example,may be bound to a tubular nanostructure using a heterofunctional crosslinker or using other methods described herein. The antibody attached tothe tubular nanostructure may be loaded with the chemotherapy agentprior to administering the tubular nanostructure to a subject.Alternatively, the chemotherapy agent may be administered before orafter administration of the tubular nanostructure. In this instance,binding of the chemotherapy to the antibody associated with the tubularnanostructure would occur in vivo.

Alternatively, an antibody that recognizes and binds a chemotherapyagent may be a bifunctional antibody. In addition to recognizing andbinding a chemotherapy agent, the bifunctional antibody may alsorecognize and bind to a ligand on the surface of the tubularnanostructure. The antibodies within the bifunctional antibody may betwo or more intact antibodies and/or two or more antibody fragments suchas, for example, Fab, F(ab)₂ and/or F_(v) that are linked in some way toone another. The two or more antibodies may be fused by chemicalconjugation, crosslinking and/or linker moieties. For example,polypeptides may be covalently bonded to one another through functionalgroups associated with the polypeptides such as, for example, carboxylicacid or free amine groups.

Alternatively, one or more antibodies may be linked through disulfidebonds. For example, the antibody that binds the chemotherapy agent maybe reacted with N-succinimidyl S-acetylthioacetate (SATA) andsubsequently deprotected by treatment with hydroxylamine to generate anSH-antibody with free sulfhydryl groups (see, e.g., U.S. Pat. App. No.2003/0215454 A1, which is incorporated herein by reference). Theantibody the binds the tubular nanostructure may be reacted withsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sSMCC). The two antibodies treated as such are purified by gelfiltration and then reacted with one another to form a bifunctionalantibody complex. Alternatively, the antibodies may be chemicallycross-linked to form a heteropolymerized complex using, for example,SPDP [N-succinimidyl-3-(2-pyridyldithio) propionate] (see, e.g., Liu, etal. PNAS 82:8648-8652, 1985; U.S. Pat. No. 5,470,570, which areincorporated herein by reference).

Alternatively, the two antibody binding activities may be incorporatedinto a single fusion protein using recombinant DNA approaches (see,e.g., U.S. Pat. No. 6,132,992, which is incorporated herein byreference). For example, cDNA encoding the variable regions (V_(L) andV_(H)) of two antibodies directed against separate and distinctantigens, for example, may be combined into a linear expressionconstruct from which a bispecific single-chain antibody may be produced(see, e.g., Haisma, et al. Cancer Gene Ther. 7:901-904, 2000, which isincorporated herein by reference). As such, cDNA encoding the variableregions (V_(L) and V_(H)) of the antibody that binds the chemotherapyagent and the antibody that binds the tubular nanostructure, forexample, may be manipulated to form a bispecific single-chain antibody.

The bifunctional antibody recognizing a chemotherapeutic agent and atubular nanostructure may be combined with the tubular nanostructureprior to administering the nanotubes to a subject. Alternatively, thebifunctional antibody may be administered before or after administeringthe tubular nanostructures. As such, binding of the bifunctionalantibody to the tubular nanostructures would occur in vivo. Thechemotherapy agent may be bound to the bifunctional antibody when thelatter is administered. Alternatively, the chemotherapy agent may beadministered separately.

Example 6

Tubular Nanostructure with Marker

One or more tubular nanostructures modified with one or more marker maybe used to selectively mark a target cell, e.g., a tumor cell. One ormore tubular nanostructures may include one or more marker that is afluorescent marker, a radioactive marker, a quantum dot, and/or magneticresonance imaging marker. The one or more tubular nanostructuresmodified with one or more marker may be selectively directed to tumorcells or other target cells through a ligand associated with the tubularnanostructures that recognizes a corresponding cognate on the targetcells. Imaging of the one or more marker may be used to monitorassociation of the tubular nanostructures with the targeted cells.

Tubular nanostructures generated using the methods described herein maybe further modified with one or more markers. For example, a tubularnanostructure that includes an antibody to the HER-2 receptor asdescribed herein may be further modified with one or more fluorescentmarkers, for example, to enable imaging of breast cancer cells. The oneor more fluorescent markers may be any of a number of fluorescent dyessome of which are described herein. For example, fluoresceinisothiocyanate (FITC) may be added to a tubular nanostructure using FITCmodified phospholipid-PEG-NH₂ (see, e.g., Kam et al., Proc. Natl. Acad.Sci. USA 102:11600-11605, 2005, which is incorporated herein byreference). PL-PEG-NH₂ may be purchased from Avanti Polar Lipids(Alabaster, Ala.) and dissolved in 0.1 M carbonate buffer solution (pH8.0) to which is added FITC (from, e.g., Sigma-Aldrich, St. Louis, Mo.).The mixture may be incubated overnight at room temperature withprotection from light. The PL-PEG-FITC may be isolated from the reactionmix by gel chromatography on a Sephadex G-25 column, for example. ThePL-PEG-FITC is mixed with carbon nanotubes and sonicated for 45 minutesto 1 hour and centrifuged at 22,000× g for 4-8 hours.

Alternatively, the one or more markers are indirectly linked to thecarbon nanotube, for example, through a fluorescently labeled protein,antibody, oligonucleotide, aptamer or combinations thereof. For example,carbon nanotubes may be modified with a commercially available antibodyto the HER-2 receptor that is itself labeled with a fluorescent marker(from, e.g, BioLegend, San Diego Calif.; R&D Systems, Inc., Minneapolis,Minn.).

In some instances, it may be beneficial to modify the tubularnanostructures with a fluorescent marker that emits at far red and/ornear infrared wavelengths to minimize interference associated withendogenous cell and tissue autofluorescence. Examples of near infraredfluorescent markers include, but are not limited to, IRDye 800CW, IRDye800RS, and IRDye 700DX (maximum emission wavelengths equal 794 nm, 786nm, and 687 nm, respectively; from LI-COR, Lincoln, Nebr.); Cy5, Cy5.5,and Cy7 (maximum emission wavelengths equal 670 nm, 694 nm, and 760 nm,respectively; from Amersham Biosciences, Piscataway, N.J.); VivoTag 680(VT680; VisEn Medical, Woburn, Mass.) and/or a variety of Alexa Fluordyes including Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, Alexa Fluor 700, and Alexa Fluor 750 (maximum emissionwavelengths equal 647 nm, 668 nm, 690 nm, 702 nm, 723 nm, and 775 nm,respectively; from Molecular Probes-Invitrogen, Carlsbad, Calif., USA;see, e.g., U.S. Pat. App. No. 2005/0171434 A1). For example, IRDye 800CWmay be added to functionalized tubular nanostructures using the methodsdescribed herein. IRDye 800CW with a reactive N-hydroxysuccinimide (NHS)group may be purchased from LI-COR, Lincoln, Nebr. The tubularnanostructures are appropriately prepared to include free amines such aswith PL-PEG-NH₂ as described above which may react with IRDye 800CW-NHSto conjugate the IRDye to the nanotube.

In vivo, non-invasive monitoring of near infrared (NIR) fluorescence,for example, may be performed using fluorescence mediated moleculartomography as described, for example, in U.S. Pat. No. 6,615,063, whichis incorporated herein by reference. Additional information regardingNIR imaging in human subjects, for example, is described in FrangioniCurr. Op. Chem. Biol. 7:626-634, 2003, which is incorporated herein byreference. In some instances, a wireless system may be used in whichlight sources such as light emitting diodes (LEDs) of appropriatewavelength as well as detectors such as charge-coupled devices (CCDs)are housed along with a power supply and a wireless communicationcircuit to create a device that may be placed on the skin of a subjectto monitor NIR signal as described by Muehlemann, et al., OpticsExpress, 16:10323, 2008, which is incorporated herein by reference.

Example 7

Tubular Nanostructure with Activatable Marker

One or more tubular nanostructures modified with one or more activatablemarker may be used to selectively mark a target cell. One or moremarkers associated with the tubular nanostructure may be activated by aligand reaction, anchoring in the membrane and interaction with ahydrophobic medium, and/or change in the cellular environment (e.g.,changes in pH). The one or more tubular nanostructures modified with oneor more marker may be selectively directed to tumor cells or othertarget cells through a ligand associated with the tubular nanostructuresthat recognizes a corresponding cognate on the target cells. Imaging ofthe one or more marker may be used to monitor association of the tubularnanostructures with the targeted cells.

In some instances, the marker associated with the tubular nanostructuresmay be activated by anchoring in the hydrophobic lipid membrane. Forexample, a tubular nanostructure may be labeled with one or morefluorescent markers that fluoresce in the presence of a lipidenvironment. Examples of lipid-sensitive fluorescent markers include,but are not limited to, nitrobenzoxadiazole (NBD), diphenylhexatrienepropionic acid (DHP), pyrene-labeled sn-2 acyl chains, and variousderivatives thereof. A tubular nanostructure may be modified with NBD,for example, using commercially available NBD derivatives ready forconjugation. For example, the NBD derivatives4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD fluoride), succinimidyl6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X, SE), and6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoic acid (from,e.g., Invitrogen, Carlsbad, Calif.) can be reacted with primary aminesas well as thiols, cysteines and secondary amines on the tubularnanostructures to conjugate NBD to the surface. NBD-X, SE, for example,may be added to tubular nanostructures with primary amines by combiningthe components together in a slightly basic buffer lacking primaryamines, e.g., 0.1-0.2 M sodium bicarbonate buffer at pH 8.3 andincubating for 1-2 hours, followed by size exclusion gel filtration toseparate the labeled nanotubes from the free NBD.

Alternatively, the maker associated with the tubular nanostructures maybe activated by binding to a specific ligand on the target cell. Forexample, the marker may be an aptamer based molecular beacon. In thisinstance, the fluorescence associated with the molecular beacon isquenched until the beacon interacts with its intended target. Tumortargeting aptamers, for example, may be generated against whole tumorcells and/or specific tumor targets using the SELEX method describedhere.

In some instances, an aptamer may have a fluorophore in a region of themolecule known to undergo conformational change upon binding of a targetthat leads to an increase in fluorescence intensity. An aptamer of thissort may be selected for using an in vitro selection process withfluorescently labeled aptamers (see, e.g., Jhaveri, et al. Nat. Biotech.18:1293-1297, 2000, which is incorporated herein by reference). A poolof RNA molecules is generated in which the random sequence region (45-60residues) is skewed such that one of the residues, uridine, for example,is disproportionately underrepresented. The three to four randomlyplaced uridine residues are substituted with fluorescein-12-UTP, CascadeBlue-7-UTP, Texas Red-5-UTP, and/or Rhodamine Green-5-UTP during invitro transcription. The labeled pool of RNA molecules are screenedagainst the target cells or a specific target associated with the cells.Those RNA molecules that bind with high affinity to the target cells ora specific target associated with the cells are further screened fortheir fluorescence signaling properties in response to binding thetarget cells or a specific target associated with the cells. Forexample, the baseline fluorescence intensity is measured for RNA aptamermolecules labeled with fluorescein-12-UTP (excitation maxima 494 nm,emission maxima 521 nm) or Rhodamine Green-5-UTP (excitation maxima 505nm, emission maxima 533 nm), for example, then re-measured in responseto increasing concentrations of target cells or a specific targetassociated with the cells. As such, fluorescent aptamers may be selectedthat exhibit a 100-200% increase in fluorescence intensity in responseto target binding.

An aptamer may be labeled either by direct incorporation of nucleicacids modified with fluorescent dyes or quenchers or by conjugation offluorescent dyes or quenchers to appropriately modified nucleic acids.For example, an aptamer may be labeled directly with Cy3. Thefluorophores may be attached to various chemical moieties that allow forattachment at various sites within the aptamer. For example, 3′-DABCYLCPG may be used to place DABCYL at the 3 prime terminus of the aptamerwhereas 5′-DABCYL phosphoramidite may be used to place DABSYL at the 5prime terminus of the aptamer (see, e.g., product information at GlenResearch; Sterling, Va.). DABCYL dT may be used to place DABCYL withinthe sequence. Labeling aptamers with appropriate commercially availablefluorophores may be achieved following instructions provided by therespective manufacturer. Alternatively, an aptamer-based molecularbeacon may be special ordered from a commercial source (from, e.g.,Biosearch Technologies, Inc., Novato, Calif., USA).

An aptamer may be attached to a carbon nanotube (So et al, JACS). Tweenmay be bound non-covalently to the carbon nanotube sidewalls throughhydrophobic interactions while the carboiimidazole may be covalentlyattached to the 3′-amine group of an RNA or DNA based aptamer.

In some instance, the tubular nanostructures may be modified with amarker that is an antibody that emits a signal a shift in emissionwavelength, for example, in response to interacting with a ligand on thetarget cell or organelle (see, e.g., Brennan (1999) J. Fluor.9:295-312). An antibody that exhibits a shift in fluorescent signal inresponse to binding of an antigen may be generated by labeling theantibody with a solvent-sensitive fluorophore such as dansyl chloride(5-dimethylaminonaphthalene-1-sulfonyl chloride), for example (see,e.g., Brennan (1999) J. Fluor. 9:295-312). The antibody is labeled suchthat binding of the antigen to the antibody shields the solventsensitive fluorescent label near the active binding site from thesolvent water, resulting in a 3-5 fold increase in fluorescenceintensity (see, e.g., Bright, et al. (1990) Anal. Chem. 62:1065-1069).As such, an antibody directed against a specific illicit drug and/ordrug of abuse, e.g., methamphetamine is incubated with methamphetamine(0.10 mg/ml) to block or protect the antibody/antigen binding site. Theantibody/antigen complex is non-selectively labeled with 0.1 uM dansylchloride under basic conditions of pH 8.5. The methamphetamine isremoved from the dansylated antibody. In this instance, for example,subsequent binding of methamphetamine will result in a measureableincrease in the intensity of the dansyl fluorescence at an emissionwavelength of 420 nm when excited with a wavelength of 325 nm.

The tubular nanostructures modified with one or more marker may befurther modified with one or more ligand that binds to a specificcognate on tumor cells. A ligand may be an antibody. An antibody may beconjugated to tubular nanostructures such as carbon nanotubes using asulfo-SMCC linkage as described in Example 1. Alternatively, an antibodyas well as other ligands may be conjugated to tubular nanostructures viaa biotin/avidin interaction. In this instance, the tubularnanostructures may be modified with a phospholipid PEG-biotin moiety andinteracted with an avidin labeled antibody. Biotin may be added tocarbon nanotubes by mixing the carbon nanotubes (0.1 to 1 mg) in 1 to 5ml of 166 μM DSPE-PEG(2000)-biotin (from Avanti Polar Lipids, Inc.,Alabaster, Ala.) with sonication for 10 minutes. The samples are washedtwice with water by centrifugation at 90,000× g for 15 minutes at 4° C.The supernatant may be discarded and the pellet resuspended in water andfurther centrifuged for 10 min at 16,000× g at room temperature. The top50% of the supernatant containing biotinylated carbon nanotubes is takenfor further conjugation. To prepare the antibody other ligand forconjugation, the antibody is thiolated with 2-iminothiolane as describedherein to add sulfhydryl groups to the protein. The avidin protein isactivated with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) asdescribed by the manufacturer (product #22311, Pierce Biotechnology,Rockford, Ill.). The thiolated antibody and the activated avidin may beconjugated to one another by mixing the two components at a molar ratioof 1:2 for 2 hours at room temperature with gentle shaking. Theresulting conjugate may be purified by gel filtration on a SephacrylS-300 HR column using 0.1 M phosphate buffered saline, 0.05% Tween-20,at pH 7.4. Carbon nanotubes modified with antibody are generated bymixing the biotinylated carbon nanotubes with the avidin labeledantibody in a 1:2 (wt/wt) ratio and incubated for 35 minutes at roomtemperature with gentle rocking. The mixture is centrifuged for 5minutes at 16,000× g at 4° C., the supernatant disgarded and the pelletused for treatment. Alternatively, the carbon nanotube may befunctionalized with streptavidin by non-covalent interactions and abiotinylated antibody or other ligand attached to the carbon nanotubevia the streptavidin-biotin interaction (see, e.g., Lyonnais et al,Small 4:442-446, 2008, which is incorporated herein by reference).

Example 8

Composite Tubular Nanostructure

Two or more tubular nanostructures may be configured to form higherorder assemblies or composite tubular nanostructures. A compositetubular nanostructure may comprise two or more tubular nanostructureseach including a hydrophobic surface region, each hydrophobic regionflanked by two hydrophilic surface regions configured to form a pore ina lipid bilayer membrane. Composite tubular nanostructures comprised oftwo or more tubular nanostructures may be used to create multiple poresat one or more sites in the targeted lipid bilayer. A composite tubularnanostructure may be generated by selective oxidation, sonication,and/or solubilization of carbon nanotube aggregates to generate smallerbundles of appropriate size and number. Alternatively, a compositetubular nanostructure may be generated from ordered assembly of singlecarbon nanotubes using biomolecule binding interactions, for example.Biomolecular binding interaction that might be used to bind a bead to atubular nanostructure include but are not limited to streptavidin-biotininteractions, antigen-antibody interactions, protein-proteininteractions, sense-anti sense DNA or RNA interactions, aptamer-targetinteraction, peptide-nucleic acid (PNA)-DNA or RNA interactions.

Acid oxidation and sonication may be used to generate a stable aqueoussuspension of purified single or small bundles of shortened nanotubes.Acid oxidation and sonication may also be used to introduce surfacecarboxylates on the nanotubes for chemical derivatization. As such,carbon nanotubes grown by laser ablation, for example, are refluxed forabout 36 hours in 2.5 M HNO3, subjected to sonication for 30 minutes,and then refluxed again for another 36 hours. The mixture may befiltered through a polycarbonate filter with a defined pore size rangingfrom 10 nm to 100 nm (see, e.g., GE PCTE filters, GE Osmonics Labstore,Minnetonka, Minn.) to isolate a defined size range of nanotubes.Optionally, centrifugation at 7000 rpm for 5 min, for example, may beused to remove larger un-reacted impurities from the solution. Atomicforce microscopy may be used to assess the size and dispersion of thetubular nanostructures following acid oxidation and Zeta potentialmeasurements may be used to confirm the existence of negatively chargedacidic groups on the nanotube sidewalls. (U.S. Patent Application2006/0275371 A1, which is incorporated herein by reference).Alternatively, scanning and/or transmission electron microscopy and/orRaman spectroscopy may be used to monitor disaggregation of carbonnanotubes.

In some instances, the composite tubular nanostructure may be built bycombining individual nanotubes that have been asymmetricallyfunctionalized with compatable binding biomolecules such as, forexample, biotin and streptavidin. For example, a polymer maskingtechnique may be used to asymmetrically modify the nanotube sidewall asdescribed by Qu & Dai Chem. Commun. 3859-3861, 2007, which isincorporated herein by reference. In this instance, one surface of thecarbon nanotubes is embedded in a polystyrene film. The exposed surfaceis subsequently modified. For example, carbon nanotubes previouslytreated with acid and sonication and containing carboxylate groups asjudged by Zeta potential measurements may be embedded in polystyrene.Carbodiimide and derivatives thereof may be used to convert thecarboxylate groups to primary amines. These reactive amines aresubsequently available for addition of other biomolecules. Additionalmodifications may be made while the nanotubes are embedded.Alternatively, the masking agent may be removed from the nanotubes priorto addition of other biomolecules. A masking agent such as polystyrene,for example, may be removed by treating the nanotubes with an treatedwith an organic solvent such as, for example, toluene.

The tubular nanotubes which have been asymmetrically functionalized withprimary amine groups may be further modified with biotin usingN-hydroxysuccinimide ester (NHS). Various NHS-biotin conjugates may beused for this purpose. For example, NHS-PEG4-Biotin and NHS-PEG12-Biotin(from Pierce-Thermo Scientific, Rockford, Ill.) may be used for simpleand efficient biotin labeling of primary amine groups associated with,for example, carbon nanotubes. The associated polyethylene glycol (PEG)spacer associated with these NHS derivatives may also increase thesolubility of the nanotubes. In some instances, it may be beneficial touse a biotin linker group with a cleavable disulfide bound (e.g., EZLink NHS-SS-Biotin; from Pierce-Thermo Scientific, Rockford, Ill.),allowing for the disruption of the nanotube bundle in, for example, theinterior of the cell.

To modify primary amines with NHS-PEG12-Biotin, for example, 1-10 mg ofprimary amine containing nanotubes are solubilized at a concentration of2-10 mg/ml in an aqueous buffer at pH 7.2-8.0. In this instance, thecarbon nanotubes, for example, may be concentrated in a small volume ofdimethylformamide (DMF) or dimethyl sulfoxide (DMSO) or other watermiscible solvent and added with gentle vortexing to the aqueous buffer.The NHS-PEG12-Biotin is similarly dissolved in DMF or DMSO other watermiscible solvent and added at 10-20 fold molar excess relative to thecarbon nanotubes. The NHS-PEG12-Biotin is allowed to incubate with thecarbon nanotubes for 2-3 hours on ice or for 30-45 minutes at roomtemperature. The unbound NHS-PEG12-Biotin may be removed by dialysis.

A second set of tubular nanostructures may be modified with avidin orstreptavidin and used with the biotin modified tubular nanostructures toform higher order bundles. Avidin or streptavidin may benon-specifically and non-covalently bound to the tubular nanostructuresas described above. Alternatively, avidin or streptavidin may be addedto tubular nanostructures using one or more of the various cross-linkingagents described herein. For example, SMCC (succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate) may be used tocrosslink the primary amines associated with functionalized carbonnanotubes with sulfhydryl groups associated with cysteine residues inavidin or streptavidin.

The tubular nanostructures modified with streptavidin and biotin, forexample, may be combined to form composite tubular nanostructures. Insome instances, the ratio of asymmetric labeled nanotubes to symmetriclabeled nanotubes may be controlled. For example, to form a heptamercomposite tubular nanostructure containing seven nanotubes, the ratio ofasymmetric to symmetric nanotubes may be 6:1, for example.

Example 9

Composite Tubular Nanostructure with Ligand

One or more composite tubular nanostructures may be used to selectivelytarget and kill tumor cells in a subject with cancer. One or morecomposite tubular nanostructure may be generated using the methodsdescribed. The one or more composite tubular nanostructures may beselectively directed to the tumor cells through a ligand associated withthe composite tubular nanostructures that recognizes a correspondingcognate on the membrane of the tumor cells. The one or more ligands maybe at least a portion of an antibody, antibody-coated liposome,polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein,carbohydrate, lipid, toxins, pore-forming toxins, or lectin. Methods formodifying tubular nanostructures with ligands have been describedherein. The one or more cognates on a membrane of a tumor cell may be aleast one of a protein, a carbohydrate, a glycoprotein, a glycolipid, asphingolipid, a glycerolipid, or a metabolite thereof. Once at thetargeted tumor cell, the one or more composite tubular nanostructuresmay form pores in the plasma membrane through which intracellular andextracellular components may flow. Disruption of the highly controlledbarrier function of the plasma membrane ultimately results in death ofthe targeted tumor cell.

Example 10

Tubular Nanostructures Targeted to Bacteria

One or more tubular nanostructures may be used to selectively target anddamage bacterial cells in a subject with a bacterial infection. The oneor more tubular nanostructures may be selectively directed to bacteriathrough a ligand associated with the tubular nanostructures thatrecognizes a corresponding cognate on the bacteria.

An antibody may be added to a tubular nanostructure to enable targetingof the nanotube to bacteria as described, for example, by Elkin et al(ChemBioChem 6:640-643, 2005, which is incorporated herein byreference). Tubular nanostructures, e.g., carbon nanotubes arefunctionalized with bovine serum albumin (BSA) using acarbodiimide-activated amidation reaction. Functionalization of thenanotubes with BSA renders the nanotubes more soluble in physiologicalbuffers. An antibody directed against one or more bacteria can benon-covalently absorbed by the nanotube-BSA conjugate. In a typicalprocedure, a solution of antibody (10 ug/ml) in phosphate-bufferedsaline (PBS) or other physiologically relevant buffer is added to thenanotube-BSA solution (20 mg/ml). The suspension is mixed by slowrotation at 40 rpm for 20-24 hours at room temperature, for example, andthen subjected to centrifugation at 14000× g to remove unbound antibody.The supernatant is discarded and the pelleted nanotube-BSA-antibodyconjugate is washed repeatedly with additional PBS and centrifugation.The resulting nanotube-BSA-antibody conjugate may be passed through amembrane filter (e.g., 0.2 μm) to eliminate clumped nanotubes. Othermethods for adding an antibody to tubular nanostructure may becontemplated, some methods of which are described herein.

Example 11

Tubular Nanostructure Targeted to Intracellular Organelle

One or more tubular nanostructure may be modified to allow transit ofthe nanotubes through the plasma membrane of a cell and subsequenttargeting and insertion of the nanotubes into the lipid bilayer of aninternal organelle such as, for example, mitochondria. In general,mitochondrial outer membrane permeabilization is considered the “pointof no return” during apoptosis of cells as it results in diffusion tothe cytosol of numerous proteins that normally reside in the spacebetween the outer and inner mitochondrial membranes and initiates acascade of events leading to cell death (see, e.g., Chipuk, et al., CellDeath Differ. 13:1396-1402, 2006, which is incorporated herein byreference). As such, one or more tubular nanostructures may be targetedto the outer membrane of mitochondria for insertion into and disruptionof the outer mitochondrial membrane, leading to cell death. In someinstances, the one or more tubular nanostructures may be furthermodified to target only mitochondria in cells of interest such as, forexample, tumor cells. As such, tubular nanostructures may be firsttargeted to tumor cells with in a subject, pass through the tumor cellmembrane, and target and disrupt the tumor cell mitochondria, leading totumor cell death.

The tubular nanostructures as described herein have a hydrophobicsurface region flanked by two hydrophilic surface regions for insertionand retention in a lipid bilayer. As such, tubular nanostructuresgenerated as described herein may be modified in such a manner as tomask the hydrophilic ends and allow transit through the plasma membraneof a target cell. In one embodiment, the hydrophilic ends of the tubularnanostructure are modified with a hydrophobic moiety using a chemicalbond that may be cleaved once the nanotube has passed into the cell.Examples of biologically cleavable bonds include, but are not limitedto, disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,acetals, ketals, enol ethers, enol esters, enamines and imines (see,e.g., U.S. Pat. Nos. 7,087,770, 7,098,030 and 7,348,453, which areincorporated herein by reference). Alternatively, the cleavable bond maybe a photolabile bond. Examples of hydrophobic moieties that might beadded to the ends of the tubular nanostructure include, but are notlimited to, non-polar hydrocarbon chains of various lengths. In oneaspect, the hydrophobic moiety is an ester that can be cleaved by anintracellular esterase to form a hydrophilic acid moiety and alcoholmoiety. For example, ceramides, which are long chain sphingoid baseslinked to fatty acids, may be conjugated to other compounds through anester linkage and used to transport compounds through the lipid bilayerand to release compounds inside the cell (see, e.g., Yatvin, et al.,Cell. Mol. Biol. Lett. 5:119-132, 2000, which is incorporated herein byreference). As such, tubular nanostructures may be modified withceramide or another long-chain nonpolar compound through an esterlinkage at one or both ends of the nanotube.

Alternatively, the masked tubular nanostructures may enter the cell bypassing directly through the cell membrane and into the cytoplasm. Inthis instance, the tubular nanostructure may include moieties on thesurface of the nanotubes that confers direct passage through the lipidbilayer, e.g., an amphiphilic striated surface on the nanotube. Thedeposition of a hydrophilic-hydrophobic striated pattern of molecules,e.g., the anionic ligand 11-mercapto-1-undecanesulphonate (MUS) and thehydrophobic ligand 1-octanethiol (OT) on the surface of nanotubes mayfacilitate direct passage of the tubular nanostructures into thecytoplasm (see, e.g., Verma, et al., Nature Materials_7: 588-95, 2008,which is incorporated herein by reference). For example, the hydrophilicends of the tubular nanostructure may be modified with an amphipathic orhydrophobic moiety using a chemical bond that may be cleaved once thenanotube has passed into the cell. Examples of biologically cleavablebonds are discussed above. Once the masked tubular nanostructures hasentered the cytoplasm, it can be modified to reveal tubularnanostructures with hydrophobic surface region flanked by twohydrophilic surface regions and at least one ligand bound to thenanostructure and configured to bind one or more cognates on anorganellar membrane, e.g., a mitochondrial membrane.

In one aspect, hydrophilic moieties may be masked by acetoxymethylesters of phosphates, sulfates, or other compounds having alcoholmoieties or acid moieties, which will enhance permeability of thetubular nanostructure across the lipid bilayer membrane. Becauseacetoxymethyl esters are rapidly cleaved intracellularly, theyfacilitate the delivery of tubular nanostructures into the cytoplasm ofthe cell without puncturing or disruption of the cell plasma membrane(see, e.g., Schultz et al., J. Biol. Chem. 268: 6316-6322, 1993, whichare incorporated herein by reference). Once within the cytoplasm, thetubular nanostructures having a hydrophobic surface region flanked bytwo hydrophilic surface regions is configured to form a pore in thelipid bilayer membrane of the cellular organelle. The cellular organellemay be mitochondria. Disruption of the outer membrane of themitrochondria by the tubular nanostructures will cause cell death.

Under certain conditions, the masked tubular nanostructures may beactively taken up by the cell through the process of endocytosis (see,e.g., Kam, et al., Angew. Chem. Int. Ed. 44:1-6, 2005, which isincorporated herein by reference). As such, the tubular nanostructuremay be optionally modified with an element that facilitates release ofthe tubular nanostructure from the endosome. For example, the maskedtubular nanostructures may be modified with all or part of the influenzavirus hemagglutinin-2 subunit (HA-2). HA-2 is a pH-dependent fusogenicpeptide that induces lysis of membranes at low pH and may be used toinduce efficient release of encapsulated material from cellularendosomes (see, e.g., Yoshikawa, et al., J. Mol. Biol. 380:777-782,2008, which is incorporated herein by reference). All or part of HA-2may be generated using standard recombinant molecular biology techniquesand attached to the tubular nanostructures using methods describedherein.

The tubular nanostructures are further modified with one or more ligandsthat binds to one or more cognates on mitochondria. The one or moreligands may be an antibody, antibody-coated liposome, polynucleotide,polypeptide, receptor, viral plasmid, polymer, protein, toxin, lectin,or any combination thereof as described herein. Cognates associated witha mitochondrial membrane may include at least one of a protein, acarbohydrate, a glycoprotein, a glycolipid, a sphingolipid, aglycerolipid, or metabolites thereof. Examples of cognates associatedwith the mitochondrial outer membrane, for example, include, but are notlimited to, carnitine palmitoyl transferase 2, translocase of outermembrane (TOM70), sorting/assembly machinery, ANT, voltage dependentanion channel (VDAC/Porin), and monoamine oxidase.

The tubular nanostructures may be modified with one or more ligands thatrecognize VDAC/Porin, for example, a common protein expressed on thesurface of the mitochondrial outer membrane. The ligand may be anantibody. Antibodies to VDAC/Porin, for example, may be generated usingstandard methods. Alternatively, antibodies to VDAC/Porin may beavailable from one or more commercial sources (from, e.g., GeneTex,Inc., San Antonio, Tex.; Sigma Aldrich, Saint Louis, Mo.; Genway BiotechInc., San Diego, Calif.). An antibody to an outer mitochondrial membranecognate such as VDAC/Porin may be attached to a tubular nanostructureusing methods described herein.

Alternatively, the ligand may be all or part of an endogenous proteinthat is binding partner of VDAC/Porin. Examples of proteins thatinteract with VDAC/Porin include but are not limited to hexokinse,glycerol kinase, and Bax (see, e.g., Vyssokikh & Brdiczka, ActaBiochimica Polonica 50:389-404, 2003, which is incorporated herein byreference). As such, all or part of hexokinase, for example, may begenerated using standard recombinant molecular biology techniques andthe known polynucleotide sequences of hexokinase available in GenBank aspart of the National Center for Biotechnology Information (NCBI) (see,e.g., Benson, et al., Nucleic Acids Res. 36:D25-D30, 2008, which isincorporated herein by reference). A protein or binding partner thatinteracts with one or more outer membrane proteins may be attached to atubular nanostructure through amine groups associated with the protein,for example, using the methods described herein.

The tubular nanostructures may be further modified with one or moreligands that targets the tubular nanostructures specifically to tumorcells. The one or more ligand may be an antibody, an aptamer and or apeptide, for example, and attached to the tubular nanostructures asdescribed here in.

Each recited range includes all combinations and sub-combinations ofranges, as well as specific numerals contained therein.

All publications and patent applications cited in this specification areherein incorporated by reference to the extent not inconsistent with thedescription herein and for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference for all purposes.

Those having ordinary skill in the art will recognize that the state ofthe art has progressed to the point where there is little distinctionleft between hardware and software implementations of aspects ofsystems; the use of hardware or software is generally (but not always,in that in certain contexts the choice between hardware and software canbecome significant) a design choice representing cost vs. efficiencytradeoffs. Those having ordinary skill in the art will appreciate thatthere are various vehicles by which processes and/or systems and/orother technologies described herein can be effected (e.g., hardware,software, and/or firmware), and that the preferred vehicle will varywith the context in which the processes and/or systems and/or othertechnologies are deployed. For example, if an implementer determinesthat speed and accuracy are paramount, the implementer may opt for amainly hardware and/or firmware vehicle; alternatively, if flexibilityis paramount, the implementer may opt for a mainly softwareimplementation; or, yet again alternatively, the implementer may opt forsome combination of hardware, software, and/or firmware. Hence, thereare several possible vehicles by which the processes and/or devicesand/or other technologies described herein may be effected, none ofwhich is inherently superior to the other in that any vehicle to beutilized is a choice dependent upon the context in which the vehiclewill be deployed and the specific concerns (e.g., speed, flexibility, orpredictability) of the implementer, any of which may vary. Those skilledin the art will recognize that optical aspects of implementations willtypically employ optically-oriented hardware, software, and or firmware.

In a general sense, those skilled in the art will recognize that thevarious aspects described herein which can be implemented, individuallyand/or collectively, by a wide range of hardware, software, firmware, orany combination thereof can be viewed as being composed of various typesof “electrical circuitry.” Consequently, as used herein “electricalcircuitry” includes, but is not limited to, electrical circuitry havingat least one discrete electrical circuit, electrical circuitry having atleast one integrated circuit, electrical circuitry having at least oneapplication specific integrated circuit, electrical circuitry forming ageneral purpose computing device configured by a computer program (e.g.,a general purpose computer configured by a computer program which atleast partially carries out processes and/or devices described herein,or a microprocessor configured by a computer program which at leastpartially carries out processes and/or devices described herein),electrical circuitry forming a memory device (e.g., forms of randomaccess memory), and/or electrical circuitry forming a communicationsdevice (e.g., a modem, communications switch, or optical-electricalequipment). Those having ordinary skill in the art will recognize thatthe subject matter described herein may be implemented in an analog ordigital fashion or some combination thereof.

The herein described components (e.g., steps), devices, and objects andthe description accompanying them are used as examples for the sake ofconceptual clarity and that various configuration modifications usingthe disclosure provided herein are within the skill of those in the art.Consequently, as used herein, the specific exemplars set forth and theaccompanying description are intended to be representative of their moregeneral classes. In general, use of any specific exemplar herein is alsointended to be representative of its class, and the non-inclusion ofsuch specific components (e.g., steps), devices, and objects hereinshould not be taken as indicating that limitation is desired.

With respect to the use of substantially any plural or singular termsherein, those having skill in the art can translate from the plural tothe singular or from the singular to the plural as is appropriate to thecontext or application. The various singular/plural permutations are notexpressly set forth herein for sake of clarity.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. It is to be understood that such depicted architectures aremerely exemplary, and that in fact many other architectures can beimplemented which achieve the same functionality. In a conceptual sense,any arrangement of components to achieve the same functionality iseffectively “associated” such that the desired functionality isachieved. Hence, any two components herein combined to achieve aparticular functionality can be seen as “associated with” each othersuch that the desired functionality is achieved, irrespective ofarchitectures or intermedial components. Likewise, any two components soassociated can also be viewed as being “operably connected,” or“operably coupled,” to each other to achieve the desired functionality,and any two components capable of being so associated can also be viewedas being “operably couplable,” to each other to achieve the desiredfunctionality. Specific examples of operably couplable include but arenot limited to physically mateable or physically interacting componentsor wirelessly interactable or wirelessly interacting components orlogically interacting or logically interactable components.

While particular aspects of the present subject matter described hereinhave been shown and described, it will be apparent to those skilled inthe art that, based upon the teachings herein, changes and modificationsmay be made without departing from the subject matter described hereinand its broader aspects and, therefore, the appended claims are toencompass within their scope all such changes and modifications as arewithin the true spirit and scope of the subject matter described herein.Furthermore, it is to be understood that the invention is defined by theappended claims. It will be understood that, in general, terms usedherein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood that if a specific number of anintroduced claim recitation is intended, such an intent will beexplicitly recited in the claim, and in the absence of such recitationno such intent is present. For example, as an aid to understanding, thefollowing appended claims may contain usage of the introductory phrases“at least one” and “one or more” to introduce claim recitations.However, the use of such phrases should not be construed to imply thatthe introduction of a claim recitation by the indefinite articles “a” or“an” limits any particular claim containing such introduced claimrecitation to inventions containing only one such recitation, even whenthe same claim includes the introductory phrases “one or more” or “atleast one” and indefinite articles such as “a” or “an”; the same holdstrue for the use of definite articles used to introduce claimrecitations. In addition, even if a specific number of an introducedclaim recitation is explicitly recited, such recitation should typicallybe interpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, typicallymeans at least two recitations, or two or more recitations).Furthermore, in those instances where a convention analogous to “atleast one of A, B, and C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, and C”would include but not be limited to systems that have A alone, B alone,C alone, A and B together, A and C together, B and C together, or A, B,and C together, etc.). In those instances where a convention analogousto “at least one of A, B, or C, etc.” is used, in general such aconstruction is intended in the sense one having skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, B,or C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together, orA, B, and C together, etc.). Virtually any disjunctive word and/orphrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A tubular nanostructure comprising: a hydrophobic surface region flanked by two hydrophilic surface regions configured to form a pore in a lipid bilayer membrane of a cell, at least one cell type specific ligand attached to the tubular nanostructure and configured to bind one or more cell type specific cognates on the membrane, and a binding moiety associated with one or both ends of the tubular nanostructure, the binding moiety having a structure to reversibly bind an agent, wherein controlled dissociation of the agent from the binding moiety results in passage of the agent through the pore formed by the tubular nanostructure in the lipid bilayer of the cell, and wherein the agent includes at least one of a therapeutic agent or a toxin.
 2. The nanostructure of claim 1, wherein the tubular nanostructure includes one or more of a carbon nanotube, cyclic peptide nanotube, crown ether nanotube, polymer nanotube, polymer/carbon nanotube, DNA nanotube, or inorganic nanotube.
 3. The nanostructure of claim 1, wherein the hydrophilic surface region includes one or more of amines, amides, charged or polar amino acids, alcohols, carboxylic groups, oxides, ester groups, ether groups, or ester-ether groups, ketones, aldehydes, or derivatives thereof.
 4. The nanostructure of claim 1, wherein the at least one cell type specific ligand is configured to bind one or more cell surface receptors or cell surface markers in the lipid bilayer membrane of the cell.
 5. The nanostructure of claim 1, wherein the at least one cell type specific ligand is configured to bind at least one of a protein, a carbohydrate, a glycoprotein, a glycolipid, a sphingolipid, a glycerolipid or a metabolite thereof.
 6. The nanostructure of claim 1, wherein one or both of the hydrophilic surface regions are at the end of the tubular nanostructure.
 7. The nanostructure of claim 1, having a length of about 1 nm to about 1500 nm.
 8. The nanostructure of claim 1, having a diameter of about 0.5 nm to about 5 nm.
 9. The nanostructure of claim 7, having a length of about 20 Å to about 40 Å.
 10. The nanostructure of claim 8, having a diameter of about 5 Å to about 20 Å.
 11. The nanostructure of claim 1, wherein the at least one cell type specific ligand includes at least a portion of an antibody, antibody-coated liposome, polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein, small chemical compound, carbohydrate, lipid, toxin, pore-forming toxin, or lectin.
 12. The nanostructure of claim 11, wherein the at least one cell type specific ligand is a therapeutic compound configured to affect a cell or process or to treat at least one of a disease, condition, or symptom.
 13. The nanostructure of claim 1, further comprising at least one second cell type specific ligand configured to bind one or more cell type specific cognates on the lipid bilayer membrane of the cell.
 14. The nanostructure of claim 1, wherein the tubular nanostructure induces cell death.
 15. The nanostructure of claim 1, further comprising a constitutively activatable marker attached to the tubular nanostructure.
 16. The nanostructure of claim 15, wherein the constitutively activatable marker is an activatable fluorescent marker having a chemical structure that constitutively fluoresces in response to a lipid environment.
 17. The nanostructure of claim 15, wherein the constitutively activatable marker is a molecular beacon having a chemical structure that constitutively fluoresces in response to interacting with a target on the membrane.
 18. The nanostructure of claim 15, wherein the constitutively activatable marker is a pH sensitive fluorescent dye.
 19. The nanostructure of claim 1, wherein the hydrophobic surface region is extended in diameter.
 20. The nanostructure of claim 19, wherein the hydrophobic surface region is extended in diameter by a disk, a stub, or a graphene sheet.
 21. A composite tubular nanostructure comprising: two or more nanotubes wherein at least one nanotube includes a hydrophobic surface region, each hydrophobic surface region flanked by two hydrophilic surface regions configured to form a pore in a lipid bilayer membrane of a cell; at least one cell type specific ligand attached to the tubular nanostructure and configured to bind one or more cell type specific cognates on the lipid bilayer membrane of the cell; a binding moiety associated with one or both ends of the at least one nanotube, the binding moiety having a structure to reversibly bind an agent, wherein controlled dissociation of the agent from the binding moiety results in passage of the agent through the pore formed by the at least one nanotube, and wherein the agent includes at least one of a therapeutic agent or a toxin; and one or more active transport polypeptide elements configured to reversibly block the pore to control transport of molecules through the tubular nanostructure.
 22. The composite tubular nanostructure of claim 21 including three or more nanotubes.
 23. The composite tubular nanostructure of claim 22, wherein at least one nanotube includes a completely hydrophobic surface region.
 24. The composite tubular nanostructure of claim 23, wherein the at least one nanotube including the completely hydrophobic surface region is surrounded by at least six nanotubes including the hydrophobic surface region flanked by two hydrophilic surface regions configured to form the pore in the lipid bilayer membrane.
 25. The composite tubular nanostructure of claim 21, wherein at least two of the nanotubes have different diameters.
 26. The composite tubular nanostructure of claim 21, wherein at least two of the nanotubes have different lengths.
 27. The composite tubular nanostructure of claim 21, wherein at least two of the two or more nanotubes are substantially parallel.
 28. The composite tubular nanostructure of claim 21, wherein at least two of the two or more nanotubes are substantially orthogonal.
 29. The composite tubular nanostructure of claim 21, wherein the hydrophobic surface region includes a single wall carbon nanotube surface region.
 30. The composite tubular nanostructure of claim 21, wherein the at least one cell type specific ligand is configured to bind one or more cell surface receptors or cell surface markers in the lipid bilayer membrane of the cell.
 31. The composite tubular nanostructure of claim 21, wherein one or both of the hydrophilic surface regions are at the end of the one or more nanotubes.
 32. The composite tubular nanostructure of claim 21, wherein the at least one cell type specific ligand is at least a portion of an antibody, antibody-coated liposome, polynucleotide, polypeptide, receptor, viral plasmid, polymer, protein, carbohydrate, lipid, toxins, pore-forming toxins, or lectin.
 33. The composite tubular nanostructure of claim 21, wherein two or more cell type specific ligands are configured to bind to the one or more cell type specific cognates on the lipid bilayer membrane of the cell.
 34. The composite tubular nanostructure of claim 21, further comprising a cytotoxic agent or antimicrobial agent.
 35. The composite tubular nanostructure of claim 21, wherein the one or more active transport polypeptide elements are on the extracellular end of the nanostructure.
 36. The composite tubular nanostructure of claim 21, wherein the one or more active transport polypeptide elements are on the cytoplasmic end of the nanostructure.
 37. The composite tubular nanostructure of claim 21, further comprising a marker attached to the nanostructure, wherein the marker includes a fluorescent marker, a radioactive marker, quantum dot, metal, or magnetic resonance imaging marker.
 38. The nanostructure of claim 1, wherein the binding moiety is a bifunctional antibody having a first portion and a second portion, the first portion capable of binding at one or both ends of the tubular nanostructure and the second portion capable of reversibly binding to the agent.
 39. The nanostructure of claim 1, further comprising one or more elements associated with one or both ends of the tubular nanostructure, the one or more elements having a size and a structure configured to physically and reversibly block the pore formed by the tubular nanostructure in the lipid bilayer membrane of the cell.
 40. The nanostructure of claim 39, wherein the one or more elements associated with one or both ends of the tubular nanostructure include one or more nanoparticles associated with one or both ends of the tubular nanostructure, the one or more nanoparticles having a size and shape to physically and reversibly block the pore formed by the tubular nanostructure in the lipid bilayer of the cell.
 41. The nanostructure of claim 40, wherein the one or more nanoparticles associated with one or both ends of the tubular nanostructure include one or more microbeads associated with one or both ends of the tubular nanostructure, the one or more microbeads having a size and shape to physically and reversibly block the pore formed by the tubular nanostructure in the lipid bilayer of the cell.
 42. The nanostructure of claim 39, wherein the one or more elements associated with one or both ends of the tubular nanostructure include one or more microbeads tethered to one or both ends of the tubular nanostructure and having a size and shape to physically and reversibly block the pore formed by the tubular nanostructure in the lipid bilayer of the cell.
 43. The nanostructure of claim 39, wherein the one or more elements associated with one or both ends of the tubular nanostructure include one or more magnetic nanoparticles associated with one or both ends of the tubular nanostructure, the one or more magnetic nanoparticles having a size and shape to physically and reversibly block the pore formed by the tubular nanostructure in the lipid bilayer of the cell in response to a magnetic field. 